UNIVERSIDADE FEDERAL DA BAHIA FACULDADE DE MEDICINA FUNDAÇÃO OSWALDO CRUZ CENTRO DE PESQUISAS GONÇALO MONIZ CURSO DE PÓS-GRADUAÇÃO EM PATOLOGIA TESE DE DOUTORADO CORPÚSCULOS LIPÍDICOS E EICOSANOIDES NOS MOMENTOS INICIAIS DA INFECÇÃO COM Leishmania infantum chagasi Théo de Araújo Santos Salvador-Ba 2013 UNIVERSIDADE FEDERAL DA BAHIA FACULDADE DE MEDICINA FUNDAÇÃO OSWALDO CRUZ CENTRO DE PESQUISAS GONÇALO MONIZ CURSO DE PÓS-GRADUAÇÃO EM PATOLOGIA CORPÚSCULOS LIPÍDICOS E EICOSANOIDES NOS MOMENTOS INICIAIS DA INFECÇÃO COM Leishmania infantum chagasi Théo de Araújo Santos Orientadora: Dra. Valéria de Matos Borges Co-orientadora: Dra. Patrícia Torres Bozza Tese apresentada ao Colegiado do Curso de Pósgraduação em Patologia como requisito para obtenção do grau de Doutor em Patologia Experimental. Salvador – Bahia – Brasil 2013 Ficha Catalográfica elaborada pela Biblioteca do Centro de Pesquisas Gonçalo Moniz / FIOCRUZ - Salvador - Bahia. S237c Araújo-Santos, Théo Corpúsculos lipídicos e eicosanoides nos momentos iniciais da infecção com Leishmania infantum chagasi . [manuscrito] / Théo de Araújo Santos. - 2013. 138 f.; 30 cm Datilografado (fotocópia). Tese (Doutorado) – Universidade Federal da Bahia - Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Pós-Graduação em Patologia Experimental, 2013 Orientadora: Drª. Valéria de Matos Borges, Laboratório Integrado de Microbiologia e Imunorregulação. Co-Orientadora: Drª. Patrícia T. Bozza, Laboratório de imunofarmacologia, IOC. 1. Corpúsculo lipídicos. 2. Eicosanoides. 3. Leishmania 4. Lutzomyia longipalpis I. Título. CDU 591.131.3:616.993.161 ii Dedico este trabalho a Carla, minha amada companheira Letícia, meu tesouro amado Meus pais Virgínia e Edielson, sempre presentes pelo exemplo Lia e João, meus queridos irmãos Cláudio Emanuel e Dona Del, meus pais postiços A Deus que está acima de todas as coisas e sempre será meu eterno amigo e companheiro iii AGRADECIMENTOS À minha orientadora Valéria de Matos Borges pela paciência, dedicação, confiança e amizade durante os últimos sete anos de minha formação acadêmica; À minha co-orientadora Patrícia T. Bozza pela inspiração e discussão construtivas nos últimos anos; À minha orientadora de doutorado SWE pela dedicação e pelas discussões científicas frutíferas e produtivas; À Sara de Moura Pontes pela dedicação nos experimentos e companheirismo durante todos esses anos de doutorado; À Elze Leite, Andrezza Souza, Elaine Arruda, Jorge Tolentino e Natali Alexandrino pelo apoio administrativo e logístico; À Deboraci Prates, Bruno Bezerril, Petter Entringer, Nívea Farias, Jaqueline Costa, Claudia Bordskyn, Natália Machado, Lilian Afonso pela amizade e pelas valiosas discussões e colaborações; À Adriana Lanfredi, Claudio Figueira, Diego Menezes e Marcos André Vannier pelo avanço em minha compreensão sobre microscopia eletrônica de transmissão; Aos professores do CPqGM, em especial aos professores Manoel e Aldina Barral pelos exemplos de dedição e aspiração científica; Aos amigos da família LIMI-LIP e do CPQGM/FIOCRUZ; To colaborators from University of Iowa – Leishmania Laboratory; Aos funcionários do biotério pelo cuidado e fornecimento dos animais; Aos funcionários da Biblioteca do CPqGM pela ajuda na busca pelas bibliografias; Ao CNPq, CPqGM e UFBA pelo suporte financeiro; A todos aqueles que contribuíram pela execução deste trabalho; Muito obrigado! iv SUMÁRIO RESUMO vi ABSTRACT vii LISTA DE ABREVIATURAS viii LISTA DE FIGURAS xi 1. INTRODUÇÃO 12 1.1. Aspectos gerais da leishmaniose visceral 12 1.2. Ciclo biológico da Leishmania 14 1.3. Papel da saliva do vetor durante os estágios iniciais da infecção por Leishmania 16 1.4. Eicosanoides na resposta inflamatória 19 1.5. Corpúsculos lipídicos e a síntese de eicosanoides 23 1.6. Corpúsculos e mediadores lipídicos na infecção por Leishmania 26 1.7. Eicosanoides e corpúsculos lipídicos de Leishmania 28 2. JUSTIFICATIVA 29 3. OBJETIVOS 30 3.1 Geral 30 3.2 Específicos 30 4. MANUSCRITOS 31 4.1 MANUSCRITO I - Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages 31 4.2 MANUSCRITO II - New Insights on the Inflammatory Role of Lutzomyia longipalpis Saliva in Leishmaniasis 43 v 4.3 MANUSCRITO III - Lutzomyia longipalpis Saliva Favors Leishmania infantum chagasi Infection Through Modulation of Eicosanoids 55 4.4 MANUSCRITO IV - Prostaglandin F2α Production in Lipid Bodies from Leishmania infantum chagasi is a Critical Virulence Factor 74 5. DISCUSSÃO 108 6. CONCLUSÕES 117 7. REFERÊNCIAS BIBLIOGRÁFICAS 118 8. ANEXO 127 9. APÊNDICE 128 vi RESUMO ARAÚJO-SANTOS, THÉO. CORPÚSCULOS LIPÍDICOS E EICOSANOIDES NOS MOMENTOS INICIAIS DA INFECÇÃO COM Leishmania infantum chagasi. Tese (Doutorado) – Centro de Pesquisas Gonçalo Moniz, Salvador, Bahia, 2013. Corpúsculos lipídicos são organelas citoplasmáticas envolvidas na produção de eicosanoides em leucócitos. Eicosanoides como as prostaglandinas têm sido envolvidos no controle da resposta inflamatória e imunológica. A saliva de Lutzomyia longipalpis participa do estabelecimento e desenvolvimento da doença pela modulação das respostas hemostática, imunológica e inflamatória do hospedeiro favorecendo a infecção. Entretanto, o papel dos eicosanoides nos momentos iniciais da infecção por Leishmania ainda não foi esclarecido, assim como a participação da saliva neste contexto. Aqui, nós investigamos o papel dos eicosanoides induzidos pela saliva de L. longipalpis e produzidos pela Leishmania infantum chagasi na infecção. O sonicado de glândula salivar (SGS) de L. longipalis induziu um aumento no número de CLs em macrófagos de maneira dose e tempo dependente, o qual esteve correlacionado com o aumento de PGE2 nos sobrenadante de cultura. As enzimas COX2 e PGE-sintase foram co-localizadas nos CLs induzidos pela saliva e a produção de PGE2 foi reduzida pelo tratamento com NS-398, um inibidor de COX-2. Nós verificamos que o SGS rapidamente estimulou a fosforilação de ERK-1/2 e PKC-α e a inibição farmacológica dessas vias inibiu a produção de PGE2 pelos macrófagos estimulados com SGS. Em seguida, nós avaliamos o efeito da saliva de L. longipalpis sobre a produção de eicosanoides durante a infecção por L. i. chagasi no modelo peritoneal murino. Nós observamos que a saliva aumentou a viabilidade intracelular de L. i. chagasi tanto em neutrófilos como em neutrófilos recrutados para a cavidade peritoneal. As células recrutadas para cavidade peritoneal apresentaram maiores níveis da relação PGE2/LTB4 e o pré-tratamento com NS-398 reverteu o efeito da saliva sobre a viabilidade intracelular dos parasitas. Parasitas como Leishmania são capazes de produzir PGs utilizando uma maquinaria enzimática própria. Neste estudo nós descrevemos a dinâmica de formação e a distribuição celular dos CLs em L. i. chagasi bem como a participação desta organela na produção de PGs. A quantidade de CLs aumentou durante a metaciclogênese assim como a expressão de PGF2α sintase (PGFS), sendo esta enzima co-localizada nos CLs. A adição de ácido araquidônico AA à cultura de L. i. chagasi aumentou a quantidade de CLs por parasita, bem como a secreção de PGF2α. A infecção com as diferentes formas de L. i. chagasi não foi capaz de estimular a formação de CLs na célula hospedeira. Por outro lado, os parasitas intracelulares apresentaram maiores quantidades de CLs. A infecção estimulou uma rápida expressão de COX-2, mas não foi detectado aumento na produção de PGF2α nos sobrenadantes. Por fim, nós verificamos a presença do receptor de PGF2α (FP) nos vacúolos parasitóforos de macrófagos infectados com L. i. chagasi. O prétratamento das células com um antagonista do receptor FP inibiu os índices de infecção de forma dose-dependente. Em conjunto, nossos dados apontam que os eicosanoides desempenham um papel crucial para evasão da resposta imune durante os momentos iniciais da infecção por L. i. chagasi com diferentes contribuições do parasita, do vetor e da célula hospedeira neste contexto. Palavras-Chave: Corpúsculos Lipídicos; Eicosanoides; Leishmania; Lutzomyia longipalpis; Saliva. vii ABSTRACT ARAÚJO-SANTOS, THÉO. LIPID BODIES AND EICOSANOIDS IN THE EARLY STEPS OF Leishmania infantum chagasi INFECTION. Tese (Doutorado) – Centro de Pesquisas Gonçalo Moniz, Salvador, Bahia, 2013. Lipid bodies (LB) are cytoplasmic organelles involved in eicosanoid production in leukocytes. Eicosanoids as prostaglandins (PG) have been implicated in the inflammatory and immune response control. Sand fly saliva participates of the establishment and development of the disease by modulation of haemostatic, inflammatory and immunological response of the host favoring the infection. However, the role of eicosanoids in the early steps of the infection remains to be investigated as well as the role of the sand fly in this context. Herein, we investigated the role of eicosanoids trigged by L. longipalpis saliva and produced by Leishmania infantum chagasi during infection. L. longipalpis salivary gland sonicate (SGS) induced an increase of LB number in the macrophages of a dose and time dependent manner, which was correlated with an increase of PGE2 release in the culture supernatants. Furthermore, COX-2 and PGE-synthase co-localized within the LBs induced by L. longipalpis saliva and PGE2 production was abrogated by treatment with NS-398, a COX-2 inhibitor. We verified SGS rapidly triggered ERK-1/2 and PKC-α phosphorylation, and blockage of the ERK-1/2 and PKC-α pathways inhibited the SGS effect on PGE2 production by macrophages. Next, we evaluated the effect of the L. longipalpis saliva in the eicosanoid production during L. i. chagasi in the murine peritoneal model. We observed SGS increased parasite viability inside recruited monocytes and neutrophils. In this regarding, SGS-recruited cells to peritoneal cavity displayed an increase in the levels of PGE2/LTB4 and the pre-treatment with NS-398 abrogated the sand fly saliva effect on parasite viability. Parasites as Leishmania are capable to produce PGs using enzymatic machinery itself. Parasite LBs amounts increased during metacyclogensis as well as the PGF2α synthase (PGFS) expression and this enzyme was colocalized on LBs. Exogenous addition of aracdonic acid in the Leishmania cultures increased LB number per parasite and PGF2α release. Macrophage infection with different forms of L. i. chagasi was not able to stimulate LB formation in the host cell. Notwithstanding, Leishmania infection upregulated COX-2 expression but this was not followed by PGF2α release by macrophages. We detected PGF2α receptor (FP) on the Leishmania PV surface. In addition, the pre-treatment of the host cells with a selective antagonist of FP, dramatically hampered Leishmania infection in a dose dependent manner. In set, our data point out a crucial role for eicosanoids to immune response evasion during early steps of L. i. chagasi infection with different contributions of parasite, vector and host cells in this context. Keywords: Lipid bodies; Eicosanoids; Leishmania infantum chagasi; Lutzomyia longipalpis; Saliva. viii LISTA DE ABREVIATURAS AA - Ácido Aracdônico BODIPY - 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene, sonda fluorescente utilizada para marcação de corpúsculos lipídicos. CL - Corpúsculo Lipídico COX - Ciclooxigenase CyPGs - Cis-Prostaglandinas EIA - Ensaio Imunoenzimático GPCR - Receptor acoplado a proteína G HBSS -/- - Solução Salina Balanceada de Hank sem Ca2+ e Mg2+ do inglês Hank´s Balanced Salts Solution without Ca2+ and Mg2+ HBSS +/+ - Solução Salina Balanceada de Hank com Ca2+ e Mg2+ do inglês Hank´s Balanced Salts Solution with Ca2+ and Mg2+ IFN-γ - Interferon-γ IL - Interleucina LDK - Quinase ligada a Corpúsculos Lipídicos em Trypanossoma brucei do inglês Lipid Droplet Kinase LO - Lipoxigenase LPS - Lipopolissacarídeo LSH - Leishmania LTB4 - Leucotrieno B4 LV - Leishmaniose Visceral ix MCP-1 - Proteína quimiotática de Macrófagos do inglês Monocyte Chemoattractant Protein-1 MET - Microscopia Eletrônica de Transmissão MIP - Proteína inibidora de Macrófago do inglês Macrophage Inhibitory Protein NO - Óxido Nítrico NS-398 - Meta-sulfonamida N-[ciclohenilona)] 4-nitrofenil, inibidor seletivo de COX-2 PACAP - Peptídeo de ativação da adenilato-ciclase pituitária do inglês Pituitary Adenylate Cyclase-Activating Peptide PAF - Fator de Ativação Plaquetária do inglês Platelet-Activanting Factor PGE2 - Prostaglandina E2 PGFS - Prostaglandina F2α sintase PGF2α - Prostaglandina F2α FP - Receptor da Prostaglandina F2α EP - Receptor da Prostaglandina E2 PLA2 - Fosfolipase A2 ROS - Espécies Reativas de Oxigênio SGS - Sonicado de Glândula Salivar de Lutzomyia longipalpis TGF-β - Fator de Crescimento Transformante Beta do inglês Transforming Growth Factor Beta TNF-α - Fator de Necrose Tumoral alfa do inglês Tumoral Necrosis Factor Alpha x VP - Vacúolo parasitóforo ΔMFI - Diferença da Intensidade Média de Fluorescência do inglês Difference of Media Fluorescence Intensity xi LISTA DE FIGURAS E TABELAS Figura 1. Ciclo biológico da Leishmania 15 Figura 2. Representação esquemática da cinética da resposta inflamatória 20 Figura 3. Representação esquemática das vias de produção dos principais eicosanoides 21 Tabela 1. Eicosanoides e seus respectivos receptores 23 Figura 4. Representação esquemática sobre micrografia eletrônica de um corpúsculo lipídico 24 12 1. INTRODUÇÃO 1.1. Aspectos gerais da Leishmaniose Visceral A leishmaniose é considerada uma das principais endemias do Mundo e o seu controle é uma das prioridades da Organização Mundial de Saúde. Estima-se que cerca de 2 milhões de novos casos sejam registrados a cada ano, sendo 500 mil de leishmaniose visceral (LV) (WHO 2010). A LV tem ampla distribuição, ocorrendo na África, Ásia, Europa, Oriente Médio e nas Américas. O Brasil está entre os países mais acometidos com a leishmaniose em seus variados aspectos clínicos. Na América do Sul, 90% dos casos registrados de LV estão no Brasil, onde anualmente são registrados 3.156 casos, em média, ao longo dos últimos onze anos, tendo a incidência da LV aumentado de 1,7 para 2,7 casos por 100.000 habitantes entre 1993 e 2003. Atualmente, a LV é observada em 19 dos 27 estados da federação, com aproximadamente 1.600 municípios envolvidos, sendo 77% dos casos registrados encontrados na região Nordeste (CHAPPUIS et al., 2007; COSTA, 2005). A LV tem como agentes etiológicos os parasitos Leishmania donovani e Leishmania infantum. Na América do Sul o agente etiológico é a Leishmania chagasi. O genoma das espécies L. infantum e L. chagasi é idêntico, sendo então, essa nomenclatura utilizada como sinonímia (WHO 2010). Durante esta tese utilizaremos o termo Leishmania infantum chagasi para distinguir a espécie trabalhada aqui daquela que ocorre na Europa. A LV é uma infecção crônica que apresenta altas taxas de morbidade e mortalidade em muitos países em desenvolvimento. Os sintomas mais prevalentes são febre alta, substancial perda de peso, esplenomegalia e hepatomegalia. Quando não tratada, a doença pode ter uma taxa de letalidade próxima a 100% dentro do período de 13 dois anos (WHO 2010). A resposta imune durante a LV humana é caracterizada por uma resposta mista Th1 e Th2 e linfoproliferação in vitro diminui com a gravidade da doença (WHO 2010). Altos níveis de mortalidade estão normalmente associados com uma co-infecção com HIV (WHO 2010) e/ou bactérias e hemorragia (ABDELMOULA et al., 2003; SAMPAIO et al., 2010). No Brasil, a maioria dos casos ocorre em crianças com menos de 10 anos de idade e as formas assintomáticas e moderadas da doença são mais frequentes (WHO 2010). Um trabalho recém-publicado, mostrou que a gravidade em casos pediátricos de LV está associada com altos níveis de citocinas proinflamatórias séricas (COSTA et al., 2013), entretanto o perfil de eicosanoides na doença permanece por ser estabelecido. Poucos estudos tem investigado preditores específicos de gravidade da doença. No Brasil, muitos esforços têm sido feitos para atender esta demanda e em 2006 foi proposto um manual para o tratamento de LV grave (Manual de Vigilância da Leishmaniose Visceral Grave, 2006). Recentemente um estudo propôs um escore de prognóstico para LV em crianças, o qual foi composto por seis fatores preditores de risco de morte por LV: sangramento de mucosa, icterícia, dispneia, infecções bacterianas, neutropenia e trombocitopenia (SAMPAIO et al., 2010). Entretanto, os possíveis mecanismos associados ao aumento da gravidade ainda são desconhecidos, mas aparentemente a inflamação sistêmica desempenha um papel central. A descrição de fatores específicos ligados a imunopatogênese da LV pode levar a descrição de potenciais biomarcadores para a gravidade da doença. Por sua vez, a avaliação desses biomarcadores pode favorecer o desenvolvimento de novos alvos terapêuticos e uma melhor condução clínicas dos casos. A LV humana pode ser parcialmente reproduzida no modelo experimental murino, uma vez que camundongos infectados não apresentam o desfecho letal da 14 doença. Em camundongos C57BL/6 e BALB/c, a injeção intravenosa de L. i. chagasi leva ao aumento do baço e do fígado, resultando em um aumento da carga parasitária nestes órgãos, nos quais ocorre o desenvolvimento de uma imunidade órgão-específica (LIESE; SCHLEICHER; BOGDAN, 2008). O fígado é o sítio de resolução da infecção aguda associada com o desenvolvimento de granulomas inflamatórios circundados por células de Kupffer infectadas e resistência a reinfecção. O baço, embora seja um sítio inicial para a produção da resposta imune mediada por célula, se torna um sítio de persistência da infecção com mudanças imunopatológicas associadas. O progresso da doença é caracterizado pelo imunocomprometimento do hospedeiro associado com altos níveis de TNF e IL-10 (STANLEY; ENGWERDA, 2007). O tratamento da LV é realizado pelo uso de antimoniais pentavalentes, entretanto a resistência a medicamentos tem aumentado, chegando a 50% dos casos na Índia (CHAPPUIS et al., 2007). A ausência de uma vacina eficaz contra a doença tem incentivado pesquisas por antígenos que possam ser utilizados como novos candidatos vacinais. Neste sentido, foram obtidos alguns sucessos com vacinas utilizando proteínas do parasita ou da saliva do vetor em modelos experimentais em hamsters e camundongo (GOMES et al., 2008). Entretanto, a busca por novos alvos terapêuticos ainda se faz necessária. 1.2. Ciclo biológico da Leishmania Leishmania é um parasita digenético, caracterizado por uma forma promastigota, extracelular e uma forma amastigota, intracelular. A forma promastigota é encontrada no trato intestinal de Diptera da família Psicodidae, onde passam por diversos estágios de diferenciação até chegar à forma promastigota metacíclica ou infectiva, em um processo denominado metaciclogênese (figura 1). 15 Durante o repasto sanguíneo, o flebotomíneo consegue o sangue do hospedeiro pela introdução de suas peças bucais na pele do hospedeiro vertebrado, dilacerando tecidos, rompendo capilares e criando um lago hemorrágico no qual se alimenta. Durante este processo, os flebotomíneos precisam inibir várias respostas hemostáticas do hospedeiro, tais como a ativação das cascatas de coagulação, vasoconstricção, agregação plaquetária e resposta imune (ANDRADE et al., 2005). Neste ambiente, flebotomíneos evoluíram um conjunto de componentes farmacológicos potentes com atividades redundantes e sinérgicas que subvertem a resposta fisiológica do hospedeiro favorecendo o repasto sanguíneo (ANDRADE et al., 2007). Vários estudos utilizando técnicas avançadas de análise têm sido conduzidos para identificar fatores salivares e suas atividades biológicas. Figura 1. Ciclo biológico da Leishmania (traduzido e adaptado de http://www.niaid.nih.gov/topics/leishmaniasis/pages/lifecycle). Lutzomyia longipalpis é o principal vetor da LV na América do Sul e a sua saliva tem sido extensivamente estudada. Durante a resposta inflamatória, a saliva de L. longipalpis induz o recrutamento celular, modula tanto a produção de anticorpos quanto a formação de imunocomplexos (SILVA et al., 2005; VINHAS et al., 2007), regula a atividade de linfócitos T e inibe células fagocíticas, tais como neutrófilos (Prates et al., 16 2011), células dendríticas (COSTA et al., 2004) e macrófagos (ZER et al., 2001). Entretanto, o papel da saliva na indução de eicosanoides, bem como sua associação a biogênese de corpúsculos lipídicos ainda não havia sido investigado até o presente estudo. 1.3. Papel da saliva do vetor durante os estágios iniciais da infecção por Leishmania As leishmanioses têm como vetores, dípteros pertencentes à ordem Phlebotominae, sendo os principais gêneros de importância médica Phlebotomus e o Lutzomyia, endêmicos do Velho Mundo e das Américas, respectivamente (SOARES; TURCO, 2003). No Brasil, o agente etiológico da LV é a Leishmania infantum chagasi, que é transmitida principalmente pelo flebotomíneo Lutzomyia longipalpis. Durante o repasto de fêmeas de flebotomíneos, os capilares epiteliais são lacerados formando um lago sanguíneo, onde a Leishmania é inoculada juntamente com a saliva do vetor. Componentes salivares do flebótomo afetam a atividade hemostática do hospedeiro, facilitando a formação do lago sanguíneo pela inibição da coagulação, aumento da vasodilatação e atração de leucócitos para o local da picada (CHARLAB et al., 1995; RIBEIRO, 1987). Este cenário favorece a infecção do hospedeiro vertebrado pela Leishmania (ANDRADE et al., 2005, 2007). Dentre as propriedades da saliva de L. longipalpis está a capacidade de estimular o recrutamento celular. Utilizando o modelo de bolsão inflamatório, Teixeira e cols. (2005) demonstraram experimentalmente que o sonicado de glândula salivar de L. longipalpis foi capaz de induzir um aumento no recrutamento de macrófagos após 12 horas de estímulo em camundongos BALB/c, mas não em camundongos C57BL/6. Este aumento foi correlacionado a expressão de CCL2/MCP-1 e seu receptor CCR2 17 (TEIXEIRA et al., 2005). A saliva de Phlebotomus dubosqi atrai monócitos in vitro (ANJILI et al., 1995) e a saliva de P. papatasi, não só atrai macrófagos como também favorece a infecção por Leishmania donovani nestas células, aumentando a carga parasitária (ZER et al., 2001). Além de induzirem o recrutamento de macrófagos, os componentes salivares de L. longipalpis inibem uma resposta pró-inflamatória em monócitos humanos estimulados com LPS (Costa et al., 2004). O tratamento com a saliva de L. longipalpis desabilita macrófagos estimulados com LPS à produção de citocinas como TNF-α e IL-10, ao passo que aumenta a capacidade produção de IL-6 nestas células (Costa et al., 2004). A saliva de L. longipalpis inibe a capacidade de macrófagos de apresentar antígenos de Leishmania a linfócitos T (THEODOS; TITUS, 1993). Foi demonstrado também que a saliva de P. papatasi é capaz de inibir a apresentação de antígeno e a produção de óxido nítrico em macrófagos infectados por Leishmania major, importante mecanismo microbicida no controle da infecção (BOGDAN; ROLLINGHOFF; DIEFENBACH, 2000; HALL; TITUS, 1995; THEODOS; TITUS, 1993). A saliva de L. longipalpis também foi capaz de estimular o influxo de neutrófilos no modelo peritonial murino, o qual foi aumentado durante a infecção por L. major (MONTEIRO et al., 2007). Dados do nosso grupo revelaram que a saliva de L. longipalpis induziu um rápido edema com acúmulo de neutrófilos quando inoculada intradermicamente na orelha de camundongos previamente expostos à picada natural do flebotomíneo (SILVA et al., 2005). Peters e cols. (2008) demonstraram em tempo real que a picada do Phlebotomus duboscqi foi capaz de induzir o rápido influxo de neutrófilos para o local da picada. Recentemente, o nosso grupo desmonstrou que a saliva L. longipalpis é capaz de induzir apoptose de neutrófilos relacionada com a supressão da produção de ROS (Prates et al., 2011). Além disso, nós demonstramos que 18 neutrófilos estimulados com a saliva de L. longipalpis produzem fatores quimiotáticos para neutrófilos e macrófagos (Prates et al., 2011), o que poderia contribuir para a transmissão da Leishmania após a picada. As proteínas da saliva de L. longipalpis foram purificadas e tiveram seus cDNAs descritos (ANDERSON et al., 2006). Dentre os componentes da saliva identificados que já tem atividade bem caracterizada na literatura estão: maxadilan (6,5 kDa), peptídeo com potente atividade vasodilatadora (Lerner et al., 1991; Svensjö et al., 2009); apirase (35,07 kDa), enzima com a ação anti-agregação plaquetária e antiinflamatória que hidrolisa ADP e ATP a AMP e ortofosfato; hialuronidase (42,28 kDa), enzima que auxilia na difusão de agentes farmacológicos da própria saliva na pele (CERNA; MIKES; VOLF, 2002); adenosina desaminase (52 kDa), enzima que hidrolisa a adenosina em inosina, que possui efeitos anti-inflamatórios (CHARLAB; ROWTON; RIBEIRO, 2000); adenosina e AMP, envolvidos na vasodilatação e anti-agregação plaquetária, substâncias que inibem a síntese de óxido nítrico e a função de linfócitos (KATZ et al., 2000); alfa-amilase (54,02 kDa), enzima responsável pela digestão de carboidratos (RIBEIRO; ROWTON; CHARLAB, 2000); 5’-nucleotidase (60,62 kDa), pertencente a família das apirases, essa enzima degrada AMP à adenosina, uma proteína com atividade vasodilatadora, anti-agregante plaquetária e imunossupressora (CHARLAB et al., 1999); a proteína LJM11 da família yellow exerce uma função kratagonista, ou seja atua como quelante, neste caso de amina biogênicas (XU et al., 2011); além de proteínas com função ainda desconhecida, como as proteínas da família D7 (15,5 a 36,3 kDa), apesar de estarem expressas em grande quantidade na saliva de flebotomíneos (VALENZUELA et al., 2004) e a família antígeno-5 (28,8 kDa) (VALENZUELA et al., 2001). 19 Apesar do conhecimento sobre a ação de alguns componentes da saliva de L. longipalpis, pouco é conhecido sobre o seu efeito na indução da produção de mediadores lipídicos. Apenas o maxadilan, proteína presente na saliva de L. longipalpis, foi implicado em ativar a produção de PGE2 em macrófagos murinos através de um receptor que reconhece um neuropeptídio, o PACAP. Este efeito induzido maxadilan parece estar associado com um perfil anti-inflamatório, pois concomitante à produção de PGE2 foi observado um aumento de IL-6 e IL-10 e a redução da produção de TNF-α (BOZZA et al., 1998; SOARES et al., 1998; SVENSJÖ et al., 2009). Recentemente, nós demonstramos que a saliva de L. longipalpis é capaz de beneficiar a infecção por L. i. chagasi pela indução de apoptose em neutrófilos associada com o aumento da produção de PGE2 e diminuição da produção de ROS por essas células (PRATES et al., 2011 – Ver apêndice). 1.4. Eicosanoides na resposta inflamatória Os mediadores lipídicos desempenham um papel importante nos estágios iniciais da inflamação, bem como nas etapas de resolução do processo inflamatório. Após a lesão tecidual, a produção de prostaglandinas e leucotrienos está associada ao processo de vasodilatação, aumento da permeabilidade vascular e recrutamento celular de neutrófilos, gerando uma resposta pró-inflamatória, característica dos primeiros estágios da resposta inflamatória aguda. Já nos estágios tardios, a fagocitose de neutrófilos apoptóticos por macrófagos recrutados para o sítio inflamatório induz uma mudança na categoria de mediadores lipídicos para um perfil anti-inflamatório e, consequentemente, há uma redução no influxo de células ao local da lesão associado ao processo de resolução da inflamação (figura 2) (LAWRENCE; WILLOUGHBY; GILROY, 2002). 20 Figura 2. Representação esquemática da cinética da resposta inflamatória. O painel abaixo da figura mostra os principais mediadores inflamatórios produzidos ao longo dessa cinética (adaptado de Lawrence et al., 2002). Mediadores lipídicos da inflamação são moléculas orgânicas biologicamente ativas que são liberadas no decorrer da resposta inflamatória. Os mediadores lipídicos mais estudados são os eicosanoides, uma família de metabólitos derivados da oxidação do ácido araquidônico (AA), uma molécula de 20 carbonos. O AA faz parte dos ácidos graxos que se encontram na porção sn-2 dos fosfolipídios de membrana e sua disponibilidade depende da capacidade relativa de enzimas de realizarem sua remoção ou reinserção nos fosfolipídios (BROCK; PETERS-GOLDEN, 2007). O processo de desacilação ou liberação do AA dos fosfolipídios de membrana está associado à atividade da enzima fosfolipase A2 (PLA2), a qual possui três famílias: a secretória e a citosólica, ambas dependentes de Ca2+ e a iPLA2, independente de cálcio. A PLA2 citosólica (cPLA2) está envolvida no processo de síntese de eicosanoides e sua ação 21 pode ser estimulada por uma série de estímulos exógenos, como citocinas, hormônios ou microrganismos (BROCK; PETERS-GOLDEN, 2007). O AA liberado pela estimulação da PLA2, por sua vez, pode ser metabolizado principalmente por duas classes de enzimas: as ciclooxigenases (COX) e a lipoxigenases (LO) (figura 3). Figura 3. Representação esquemática das vias de produção dos principais eicosanoides (retirado de Bozza et al. 2011). As COXs são isoenzimas que catalisam, a partir do AA, a formação de prostaglandina H2, a qual pode ser convertida pela ação de PG sintases célula-específica em diversas moléculas biologicamente ativas, tais como: PGE2, PGF2α, PGI2, PGD2 e tromboxano A2 (TXA2), coletivamente conhecidos como prostanóides (FUNK, 2001). A COX-1 tem expressão constitutiva, sendo a enzima responsável pela síntese basal de prostanóides, enquanto que a COX-2 é importante em vários processos inflamatórios 22 devido a sua expressão ser induzível (FUNK, 2001). Existe ainda a COX-3, a qual é um produto do splicing alternativo da COX-1 (CHANDRASEKHARAN et al., 2002). No contexto da infecção com microrganismos, a produção de prostaglandina E2 tem sido associada ao aumento da produção de cAMP e supressão da resposta imune do hospedeiro com a inibição da produção de citocinas pró-inflamatórias, tais como: IFN-γ, TNF-α, IL-12, IL-2 e IL-1β. Em contrapartida, a PGE2 é capaz de induzir a produção de citocinas de perfil Th2, bem como IL-10, IL-4 e imunoglobulinas do tipo IgE e IgG1 (HARRIS et al., 2002). As lipoxigenases constituem a outra via de metabolismo do AA, dentre as quais a 5- lipoxigenase (5-LO) se destaca pela produção de leucotrienos (LTs) e lipoxinas (LXs). A expressão da 5-LO está correlacionada a eventos de inflamação da fase aguda, com a produção de citocinas pró-inflamatórias e radicais de oxigênio. Entre os produtos da via da 5-LO se destacam o LTB4 em doenças infecciosas e os chamados cistenil-leucotrienos LTC4, LTD4 e LTE4, envolvidos na resposta alérgica (PetersGolden et al., 2007). O LTB4 está correlacionado com o aumento da produção de citocinas pró-inflamatórias e diminuição da infecção em diversas patologias, associado ao aumento da produção de óxido nítrico (PETERS-GOLDEN et al., 2005; ROGERIO; ANIBAL, 2012). Os eicosanoides se ligam a receptores associados à proteína G (GPCRs). A ação dos eicosanoides na resposta inflamatória está intimamente associada à cascata de transdução do sinal ativada pelos receptores aos quais eles se ligam. Dentre os eicosanoides, a PGE2 é a molécula que apresenta uma maior variedade de resposta durante a ativação por se ligar a quatro diferentes receptores: EP1, EP2, EP3 e EP4. Os eicosanoides e seus respectivos receptores, bem como o efeito da inter-relação entre estes estão listados na tabela abaixo: 23 Eicosanoide LTB4 Receptor BLT1 e 2 LTC4, LTD4, LTE4 Cys-LT1 e 2 PGF2α* FP DP1 DP2 EP1 EP3 EP2/4 IP TP PGD2 PGE2 PGI TXA2 Ativação Gqi ↑ Ca2+ ↓cAMP Gqi / Gs Gs Gqi Gi ↑ Ca2+ - ↑ cAMP ↓cAMP ↓cAMP Gs - ↑ cAMP Gq ↑ Ca2+ - Tabela 1. Eicosanoides e seus respectivos receptores. São mostrados na tabela os desfechos da ativação quanto ao tipo de proteína G ativada, produção de Ca2+ e cAMP (BOS et al., 2004; PetersGolden, 2007; Medeiros et al., 2012; PETERS-GOLDEN; HENDERSON JR.; HENDERSON, 2007). *PGF2α pode se ligar também aos receptores EP1 e EP3 (BOS et al., 2004). 1.5. Corpúsculos lipídicos e a síntese de eicosanoides Corpúsculos lipídicos (CLs) são organelas citoplasmáticas compostas de um conjunto de lipídios neutros, tais como diacilglicerol, triacilglicerol, caveolina e ésteres de colesterol circundados por uma hemi-membrana composta de fosfolipídios (BOZZA et al., 2011). Os CLs estão envolvidos no estoque e processamento de lipídios e estão presentes em todos os organismos. No entanto, apenas recentemente, os corpúsculos lipídicos foram reconhecidos como organelas (FARESE; WALTHER, 2009), uma vez que participam em diversos processos celulares como sinalização, tráfico de membranas e síntese de mediadores inflamatórios (BOZZA et al., 2011). Os CLs apresentam uma grande quantidade de AA, o principal substrato utilizado na síntese de eicosanoides. Os CLs também possuem uma grande quantidade de proteínas relacionadas com o processo de sinalização celular e endereçamento de vesículas (WAN et al., 2007). Além disso, os CLs podem apresentar enzimas 24 diretamente relacionadas à síntese de eicosanoides, as COXs e LOs (BOZZA et al., 2011). Tem sido demonstrado que os CLs podem ser os principais sítios intracelulares de produção de eicosanoides, uma vez que possuem todo o aparato enzimático e de substrato. O ambiente hidrofóbico dos CLs é ideal para o funcionamento da maquinaria responsável pela síntese de mediadores lipídicos. Foi demonstrado que a formação de CLs, sua constituição lipídica e o seu engajamento na produção de mediadores lipídicos específicos estão diretamente correlacionados ao estímulo inflamatório envolvido (figura 4). Neste sentido, a formação de CLs em leucócitos teria um importante papel durante a resposta inflamatória em diversos processos patogênicos (D’AVILA; MAYAMONTEIRO; BOZZA, 2008) Figura 4. Representação esquemática sobre micrografia eletrônica de um corpúsculo lipídico. Na imagem são ilustrados alguns aspectos moleculares da organela bem como algumas vias de sinalização envolvidas na sua formação. 25 No contexto da infecção por patógenos, tem sido mostrado que estas organelas participam ativamente da produção de mediadores durante a infecção. Pacheco e cols. (2002) mostraram que LPS é capaz de induzir a formação de CLs de maneira dose e tempo dependente e identificou nestas organelas enzimas das vias de produção de leucotrienos e prostaglandinas, o que esteve associado com a produção destes mediadores in vivo (PACHECO et al., 2002). Componentes isolados da membrana de microrganismos tais como de M. bovis aumentaram a quantidade de corpúsculos lipídicos em macrófagos, o que esteve associado com um aumento na produção de PGE2 (D’AVILA et al., 2008). Ainda neste contexto, Melo e cols. (2003) mostraram que durante a infecção em ratos por Trypanosoma cruzi houve uma intensa formação de CLs em macrófagos peritoneais, o que esteve correlacionada com a produção de PGE2 no sítio inflamatório (MELO et al., 2003; MELO; SABBAN; WELLER, 2006). Durante a infecção por T. cruzi a presença no tecido cardíaco de corpúsculos lipídicos em macrófagos infectados é um indício de ativação celular (MELO, 2008). Diferentes patógenos intracelulares se beneficiam da formação de CLs nas células hospedeiras. A formação dessas organelas e sua associação com os vacúolos parasitóforos foram demonstradas em infecções por Trypanossoma cruzi (D’AVILA et al., 2011), Toxoplasma gondii (CHARRON; SIBLEY, 2002) e Plasmodium falciparum (JACKSON et al., 2004). A distribuição dessas organelas próxima aos fagolisossomos sugere a possibilidade do corpúsculo lipídico servir como fonte de nutriente para o patógeno. Esses achados sugerem então, que a indução da formação de corpúsculos lipídicos por patógenos intracelulares pode ser uma via de inibição da resposta do hospedeiro. 26 1.6. Corpúsculos e mediadores lipídicos na infecção por Leishmania Os eicosanoides desempenham um papel crucial na infecção por Leishmania. A maioria dos estudos que investigaram a participação dos eicosanoides na leishmaniose utilizaram L. amazonensis como modelo experimental. Durante a infecção de macrófagos por L. amazonensis, PAF (LONARDONI et al., 2000) e LTB4 (SEREZANI et al., 2006) induziram a morte do parasito. Recentemente, o nosso grupo também demonstrou participação de LTB4 na morte de L. amazonensis em neutrófilos pela indução da produção de ROS e ativação da NFκB (Machado et al. 2013, manuscrito em preparação). A outra via de processamento do AA é a das COXs. Diversos trabalhos têm demonstrado que a ativação de COX beneficia a infecção por L. amazonensis pela produção de PGE2 (AFONSO et al., 2008; LONARDONI et al., 2000; PINHEIRO et al., 2008). A interação entre macrófagos humanos infectados e neutrófilos apoptóticos no modelo experimental humano (AFONSO et al., 2008) e murino (RIBEIRO-GOMES et al., 2005) resultou no sucesso da infecção por Leishmania e aumento da carga parasitária por um mecanismo de supressão da resposta imune dependente da produção de PGE2 e TGF-β. Um fator crucial para resposta induzida pelos eicosanoides é o receptor envolvido na ativação da célula hospedeira. A PGE2 pode desempenhar tanto um papel anti-inflamatório como pró-inflamatório a depender dos receptores expressos pela célula alvo (HARRIS et al., 2002). A PGE2 possui 4 receptores diferentes que são diferencialmente expressos em macrófagos, são eles EP1, 2, 3 e 4 (HARRIS et al., 2002). Os receptores EP1 e EP3 estão associados com a resposta pro-inflamatória com ativação de PKC e diminuição de cAMP, respectivamente. Já os receptores EP2 e EP4 27 estão associados à resposta anti-inflamatória, pela ativação de proteína G estimulatória com aumento dos níveis de cAMP. Recentemente, foi demonstrado que a infecção por L. major induz a expressão de EP1 e EP3 e, que a ativação desses receptores está associada com o aumento da carga parasitária, enquanto que a ativação de EP2 e EP4 induziu a redução da carga parasitária (PENKE et al., 2013). A indução da produção de PGE2 também foi demonstrada para espécies que causam leishmaniose visceral, tais como L. donovani (REINER; NG; MCMASTER, 1987) e L. infantum (MATTE et al., 2001; PANARO et al., 2001). Entretanto, o papel do PGE2 na infecção por L. infantum permanece por ser determinado. Foi demonstrado que macrófagos murinos infectados por L. donovani tem o metabolismo de AA direcionado à produção de PGE2 (REINER; MALEMUD, 1984, 1985; REINER; SCHULTZ; MALEMUD, 1988). Matte e cols. (2001) demonstraram que L. donovani é capaz de induzir a expressão de COX-2 e produção de PGE2, entretanto Panaro e cols. (2001) demonstraram que macrófagos humanos tratados com PGE2 eliminam melhor os parasitas internalizados. A infecção por L. donovani de macrófagos induziu uma maior expressão de COX e PGE sintase quando comparada a infecção por L. major, o que sugere haver a indução de respostas distintas a depender da espécie de Leishmania (GREGORY et al., 2008). Apesar de existirem vários trabalhos mostrando a importância dos eicosanoides para infecção por Leishmania, os dados sobre a formação de CLs lipídicos em células infectadas são escassos. Pinheiro e cols. (2008) mostraram que a infecção por L. amazonensis só foi capaz de induzir a formação de CLs em células de camundongos Balb/c privadas de nutrientes, e esta formação esteve associada com a produção de PGE2. Durante a infecção por L. major foi observado a formação de CLs em macrófagos derivados de medula, mas não foi observada uma produção de PGE2 28 associada a essa formação (RABHI et al., 2012). Desta forma, o papel dos CLs na infecção por Leishmania, bem como por L. i. chagasi permanece por ser estudado. 1.7. Eicosanoides e Corpúsculos lipídicos de Leishmania O estudo de CLs em diversos parasitas tem sido direcionado à participação destas organelas no estoque e metabolismo de lipídios. Em Toxoplasma gondi estas inclusões têm sido implicadas no armazenamento de lipídios “seqüestrados” da célula hospedeira, embora o mecanismo pelo qual o parasito obtém os lipídeos intracelularmente aindam não sejam bem compreendidos (NISHIKAWA et al., 2005; QUITTNAT et al., 2004). CLs também foram caracterizadas ultraestruturalmente em Leishmania donovani (CHANG, 1956). Pimenta e cols. (1991) correlacionaram o aumento do número de inclusões lipídicas em promastigotas Leishmania com o processo de metaciclogênese, produção e endereçamento de LPG à membrana plasmática do parasita (PIMENTA; SARAIVA; SACKS, 1991). O aumento dos CLs em Leishmania esteve correlacionado com o tratamento com drogas leishmanicidas que afetavam a via de síntese de ergosterol, importante componente estrutural da membrana plasmática dos parasitas (VANNIER-SANTOS et al., 1995). Apesar da semelhança morfológica entre os CLs dos leucócitos e os de células de outros organismos, a função de CLs de parasitas e a produção de eicosanoides por estes CLs ainda não foi demonstrada. Genes homólogos a COX e proteínas análogas não existem em organismos da Ordem Trypasomatidae, contudo parasitas tais como Leishmania são capazes de metabolizar ácido araquidônico a PGs (KUBATA et al., 2007). A produção de PGs por Leishmania é possível, por que estes parasitas possuem uma enzima chamada prostaglandina F2α sintase (PGFS), a qual é responsável pela 29 produção de PGF2α (KABUTUTU et al., 2003). Os sítios de produção intracelular bem como a participação dos CLs na síntese de PGF2α eram desconhecidos até o presente estudo. Além disso, não existe dado na literatura sobre a participação da PGF2α na resposta imune, o que torna este campo atraente para investigação científica. 2. JUSTIFICATIVA A saliva total e as frações proteicas de L. longipalpis têm sido cogitadas como antígenos vacinais devido à importância deste componente na transmissão por Leishmania. Apesar de existirem trabalhos na literatura sobre a importância de eicosanoides para a infecção por Leishmania, não existiam dados sobre o papel dos eicosanoides nos estágios iniciais da doença até o presente estudo. Este trabalho contribuiu neste sentido, mostrando que a saliva de Lutzomyia longipalpis é capaz de beneficiar a infecção por L. i. chagasi por modular a produção de eicosanoides. Além disso, a capacidade de produção de eicosanoides pelos parasitas e essa característica como um fator de virulência é negligenciada pela literatura. O estudo sobre os mecanismos de produção de eicosanoides por L. i. chagasi traz novas perspectivas para o entendimento da biologia celular da Leishmania e suas implicações com a célula hospedeira. 30 3. OBJETIVOS 3.1. Geral Investigar o papel dos corpúsculos lipídicos e eicosanoides produzidos durante os momentos iniciais da infecção por Leishmania infantum chagasi 3.2. Específicos Avaliar o efeito da saliva de L. longipalpis na ativação celular quanto à formação de corpúsculos lipídicos e produção de eicosanoides in vivo e in vitro; Investigar vias de sinalização celular envolvidas no processo de ativação da produção de eicosanoides induzidos pela saliva de L. longipalpis in vitro; Avaliar o efeito da saliva de L. longipalpis na produção de eicosanoides durante a infecção por L. i. chagasi in vivo e ex vivo; Investigar o envolvimento dos corpúsculos lipídicos na capacidade de produção de eicosanoides por L. i. chagasi; Avaliar a contribuição de eicosanoides produzidos pela L. i. chagasi como fator de virulência e na infecção in vitro. 31 4. MANUSCRITOS 4.1. MANUSCRITO I Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages A Saliva de Lutzomyia longipalpis Induz a Formação de Corpúsculos Lipídicos e a Produção de Prostaglandina E2 em Macrófagos Murinos Este trabalho avalia o efeito da saliva de L. longipalpis na ativação celular de macrófagos quanto à formação de corpúsculos lipídicos e a produção de eicosanoides associada a essas organelas, bem como vias de sinalização envolvidas neste processo. Resumo dos resultados: Neste estudo vimos que o sonicado de glândula salivar (SGS) de L. longipalpis induziu o recrutamento de neutrófilos e macrófagos para a cavidade peritoneal com cinética distinta para ambos os tipos celulares. A saliva do flebotomíneo induziu a produção de PGE2 e LTB4 em leucócitos após a estimulação com ionóforo de cálcio ex vivo. Após três e 6 horas de inoculada, a saliva induziu o aumento de CLs em macrófagos, mas não em neutrófilos quando comparados ao grupo controle que recebeu solução salina. Além disso, macrófagos peritoneais residentes quando estimulados com SGS in vitro tiveram um aumento no número de CLs de maneira dose e tempo dependente, o qual esteve correlacionado com o aumento de PGE2 nos sobrenadante de cultura. As enzimas COX-2 e PGE-sintase foram co-localizadas nos CLs induzidos pela saliva e a produção de PGE2 foi reduzida pelo tratamento com NS-398, um inibidor de COX-2. Por fim, nós verificamos que o SGS rapidamente estimulou a fosforilação de 32 ERK-1/2 e PKC-α e a inibição farmacológica dessas vias inibiu a produção de PGE2 induzida pela saliva. Este artigo foi publicado no periódico internacional PLoS Neglected Tropical Diseases (Fator de impacto JCR 2011 = 4.752). 33 Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages Théo Araújo-Santos1,2, Deboraci Brito Prates1,2, Bruno Bezerril Andrade1,2, Danielle Oliveira Nascimento3, Jorge Clarêncio1, Petter F. Entringer1, Alan B. Carneiro4, Mário A. C. Silva-Neto4, José Carlos Miranda1, Cláudia Ida Brodskyn1,2,5, Aldina Barral1,2,5, Patrı́cia T. Bozza3, Valéria Matos Borges1,2,5* 1 Centro de Pesquisas Gonçalo Moniz, FIOCRUZ-BA, Salvador, Brasil, 2 Universidade Federal da Bahia, Salvador, Brasil, 3 Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Rio de Janeiro, Brasil, 4 Institutos de Bioquı́mica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil, 5 Instituto de Investigação em Imunologia, Instituto Nacional de Ciência e Tecnologia (INCT), São Paulo, Brasil Abstract Background: Sand fly saliva contains molecules that modify the host’s hemostasis and immune responses. Nevertheless, the role played by this saliva in the induction of key elements of inflammatory responses, such as lipid bodies (LB, also known as lipid droplets) and eicosanoids, has been poorly investigated. LBs are cytoplasmic organelles involved in arachidonic acid metabolism that form eicosanoids in response to inflammatory stimuli. In this study, we assessed the role of salivary gland sonicate (SGS) from Lutzomyia (L.) longipalpis, a Leishmania infantum chagasi vector, in the induction of LBs and eicosanoid production by macrophages in vitro and ex vivo. Methodology/Principal Findings: Different doses of L. longipalpis SGS were injected into peritoneal cavities of C57BL/6 mice. SGS induced increased macrophage and neutrophil recruitment into the peritoneal cavity at different time points. Sand fly saliva enhanced PGE2 and LTB4 production by harvested peritoneal leukocytes after ex vivo stimulation with a calcium ionophore. At three and six hours post-injection, L. longipalpis SGS induced more intense LB staining in macrophages, but not in neutrophils, compared with mice injected with saline. Moreover, macrophages harvested by peritoneal lavage and stimulated with SGS in vitro presented a dose- and time-dependent increase in LB numbers, which was correlated with increased PGE2 production. Furthermore, COX-2 and PGE-synthase co-localized within the LBs induced by L. longipalpis saliva. PGE2 production by macrophages induced by SGS was abrogated by treatment with NS-398, a COX-2 inhibitor. Strikingly, SGS triggered ERK-1/2 and PKC-a phosphorylation, and blockage of the ERK-1/2 and PKC-a pathways inhibited the SGS effect on PGE2 production by macrophages. Conclusion: In sum, our results show that L. longipalpis saliva induces lipid body formation and PGE2 production by macrophages ex vivo and in vitro via the ERK-1/2 and PKC-a signaling pathways. This study provides new insights regarding the pharmacological mechanisms whereby L. longipalpis saliva influences the early steps of the host’s inflammatory response. Citation: Araújo-Santos T, Prates DB, Andrade BB, Nascimento DO, Clarêncio J, et al. (2010) Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages. PLoS Negl Trop Dis 4(11): e873. doi:10.1371/journal.pntd.0000873 Editor: Jesus G. Valenzuela, National Institute of Allergy and Infectious Diseases, United States of America Received June 29, 2010; Accepted October 6, 2010; Published November 2, 2010 Copyright: ß 2010 Araújo-Santos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq), Instituto de Investigação em Imunologia, Instituto Nacional de Ciência e Tecnologia (INCT) and Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB). TAS, DBP, BBA, DON, PFE and ABC received fellowships from the CNPq. VMB, PTB, CIB, AB and MACSN are senior investigators from CNPq. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] redundant and synergistic activities that subvert the host’s physiological responses and favor the blood meal. Intense research using high-throughput analyses has been conducted to identify salivary factors and their biological activities. Lutzomyia (L.) longipalpis, the main vector of visceral leishmaniasis in South America, has been extensively studied. During the inflammatory response, L. longipalpis saliva induces cellular recruitment, modulates both antibody production and the formation of immunocomplexes [3,4], regulates T cell activities and inhibits dendritic cells and macrophages, the latter being preferential host cells for Introduction To obtain a blood meal, sand flies locate blood by introducing their mouthparts into the vertebrate host’s skin, tearing tissues, lacerating capillaries and creating hemorrhagic pools upon which they feed. During this process, sand flies need to circumvent a number of the host’s homeostatic responses, such as activation of blood coagulation cascades, vasoconstriction, platelet aggregation and immune responses [1,2]. In this environment, sand flies evolved an array of potent pharmacologic components with www.plosntds.org 1 November 2010 | Volume 4 | Issue 11 | e873 Sand Fly SGS Triggers Eicosanoid Production Author Summary Methods After the injection of saliva into the host’s skin by sand flies, a transient erythematous reaction is observed, which is related to an influx of inflammatory cells and the release of various molecules that actively facilitate the blood meal. It is important to understand the specific mechanisms by which sand fly saliva manipulates the host’s inflammatory responses. Herein, we report that saliva from Lutzomyia (L.) longipalpis, a widespread Leishmania vector, induces early production of eicosanoids. Intense formation of intracellular organelles called lipid bodies (LBs) was noted within those cells that migrated to the site of saliva injection. In vitro and ex vivo, sand fly saliva was able to induce LB formation and PGE2 release by macrophages. Interestingly, PGE2 production induced by L. longipalpis saliva was dependent on intracellular mechanisms involving phosphorylation of signaling proteins such as PKC-a and ERK-1/2 and subsequent activation of cyclooxygenase-2. Thus, this study provides new insights into the pharmacological properties of sand fly saliva and opens new opportunities for intervening with the induction of the host’s inflammatory pathways by L. longipalpis bites. Antibodies and Reagents Dimethylsulfoxide (DMSO) was purchased from ACROS Organics (New Jersey, NJ). RPMI 1640 medium and L-glutamine, penicillin, and streptomycin were from Invitrogen (Carlsbad, CA). Nutridoma-SP was from Roche (Indianapolis, IN). A23187 calcium ionophore, was from Calbiochem/Novabiochem Corp. (La Jolla, CA). NS-398, PGE2 and LTB4 enzyme-linked immunoassay (EIA) Kits, anti-murine COX-2 and PGE-synthase antibodies were all from Cayman Chemical (Ann Arbor, MI). 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) was obtained from Molecular Probes (Eugene, OR). Osmium tetroxide (OsO4) was obtained from Electron Microscopy Science (Fort Washington, PA). Aqua Polymount was from Polysciences (Warrington, PA). Thiocarbohydrazide, Ca2+-Mg2+-free HBSS(2/2), HBSS(+/+) with Ca2+Mg2+, LPS from Escherichia coli (serotype 0127:b8), and N-ethyl-N’(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-mouse kinase proteins were from Santa Cruz Biotechnology (Santa Cruz, CA). PD 98059, 29-Amino-39-methoxyflavone and Bisindolylmaleimide-I, 2-[1-(3-Dimethylaminopropyl)-1H-indol-3yl]-3-(1H-indol-3-yl)-maleimide were obtained from Merck-Calbiochem (Darmstadt, Hessen). Leishmania [5,6]. There is also evidence that maxadilan, a L. longipalpis salivary protein with vasodilator properties, downregulates LPS-induced TNF-a and NO release through a mechanism dependent on PGE2 and IL-10 [7]. PGE2 is an eicosanoid derived from arachidonic acid (AA) metabolism by the enzyme cyclooxygenase (COX). Prostanoids and leukotrienes can be intensely produced by macrophages during inflammatory responses [8], and these mediators are implicated in cellular recruitment and activation. Among the eicosanoids, LTB4 induces neutrophil recruitment [9], whereas PGE2 and PGD2 attract mainly macrophages [10]. Previous studies used different experimental models to show that L. longipalpis saliva induces an influx of neutrophils [11] and macrophages [12], but neither the role of saliva in LTB4 and PGE2 release nor the involvement of these mediators in this process has been fully addressed. Under inflammatory and infectious conditions, prostaglandins and others lipid mediators are mainly produced by cytoplasmic organelles called lipid bodies (LB) [13]. Intense research over the past few years has defined lipid bodies as dynamic cytoplasmic organelles. It has been demonstrated that lipid bodies compartmentalize enzymes involved in the biosynthesis, transport and catabolism of lipids, proteins involved in membrane and vesicular transport and proteins involved in cell signaling and inflammatory mediator production, including eicosanoid-forming enzymes, phospholipases and protein kinases. All of these molecules can be localized into lipid bodies in various cells under a range of activation conditions, suggesting a wide role for lipid bodies in the regulation of cellular lipid metabolism and signaling [13]. Herein, we evaluated the effect of L. longipalpis salivary gland sonicate (SGS) on the induction of LB formation as well as PGE2 and LTB4 production in vitro and ex vivo. Moreover, we explored the role of peritoneal macrophages in the production of these lipid mediators in response to L. longipalpis SGS in vitro. Finally, we found that the PGE2 production induced by L. longipalpis saliva is dependent on intracellular mechanisms involving the phosphorylation of signaling proteins such as PKC-a and ERK-1/2 and subsequent activation of COX-2. www.plosntds.org Mice Inbred male C57BL/6 mice, age 6–8 weeks, were obtained from the animal facility of Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz (CPqGM-FIOCRUZ, Bahia, Brazil). All experimental procedures were approved and conducted according to the Animal Care and Using Committee of the FIOCRUZ. Sand flies and preparation of salivary glands Adult Lutzomyia longipalpis captured in Cavunge (Bahia, Brazil) were reared at the Laboratório de Imunoparasitologia/CPqGM/ FIOCRUZ (Bahia, Brazil) as described previously [3]. Salivary glands were dissected from 5- to 7-day-old L. longipalpis females under a Stemi 2000 Carl Zeiss stereoscopic microscope (Göttingen, Germany) and stored in groups of ten pairs in 10 mL of endotoxin-free PBS at 270uC. Immediately before use, the glands were sonicated with a Branson Sonifier 450 (Danbury, CT) and centrifuged at 10,0006 g for four minutes. The supernatant from salivary gland sonicate (SGS) was used for experiments. The level of LPS contamination of L. longipalpis SGS preparations was determined using a commercially available LAL Chromogenic Kit (Lonza Bioscience, Walkersville, MD); negligible levels of endotoxin were found in the salivary gland supernatant (0.1 gg/mL). We measured 0.7 micrograms of protein in an amount equivalent to 0.5 pair of salivary glands and used SGS dilutions (2.0–0.2 pairs) in our experiments [14]. Leukocyte recruitment to the peritoneal cavity To assess the leukocyte recruitment induced by L. longipalpis SGS, we used the well-established peritoneal model of inflammation because the peritoneal cavity is a self-contained and delineated compartment and thus provides a large number of post-stimulus leukocytes. As previously established in the air pouch murine model [12] and peritoneal cavity (unpublished data), a 0.5pair dose of SGS was used for the leukocyte recruitment assay. C57BL/6 mice were inoculated i.p. with 0.1 mL of L. longipalpis SGS (0.5 pair/cavity), endotoxin-free saline (negative control) or 0.1 mL of LPS (20 mg/mL, positive control). At 1, 3 and 6 h poststimulus, leukocytes inside the peritoneal cavity were harvested by 2 November 2010 | Volume 4 | Issue 11 | e873 34 35 Sand Fly SGS Triggers Eicosanoid Production injection and recovery of 10 mL of endotoxin-free saline. Total counts were performed on a Neubauer hemocytometer after staining with Turk’s solution. Differential cell counts (200 cells total) were carried out microscopically on cytospin preparations stained with Diff-Quick. Immunofluorescence for COX-2 and PGE-synthase Resident peritoneal macrophages were cultured on coverslips in the presence of L. longipalpis SGS (1.5 pair/well) as described above. After 24 h, the cells were washed twice with 500 ml of HBSS2/2 and immediately fixed with 500 mL of water-soluble EDAC (1% in HBSS2/2), used to cross-link eicosanoid carboxyl groups to amines in adjacent proteins. After 15 min of incubation at room temperature (RT) with EDAC to promote both cell fixation and permeabilization, macrophages were then washed with HBSS2/2 and incubated with 1 mM BODIPY 493/503 for 30 min. Then, the cover slips were washed with HBSS2/2 and incubated with mouse anti-COX-2 (1:150) or anti-PGE-synthase (1:150) for 1 h at RT. MOPC 21 (IgG1) was used as a control. After further washes, cells were incubated with biotinylated goat anti-rabbit IgG secondary Ab, washed twice and incubated with avidin conjugated with PE for 30 min. The cover slips were then washed three times and mounted in Vectashield medium containing DAPI (Vector Laboratories, Burlingame, CA). The samples were observed by fluorescence microscopy and images were acquired using the software Image-Pro Plus (Media Cybernetics, Silver Spring, MD). Lipid body staining and quantification Cells harvested by peritoneal lavage 1, 3, 6 or 24 h after i.p. injection of 0.1 mL of L. longipalpis SGS (0.5 pair/cavity), endotoxin-free saline or LPS (20 mg/mL) were centrifuged at 4006 g and the lipid bodies within the leukocytes were stained with BODIPY 493/503 (5 ug/mL) according to Plotkowisk et al. [15]. Samples were analyzed using a FACSort flow cytometer from Becton Dickinson Immunocytometry Systems (San Jose, CA) and by fluorescence microscopy. Macrophages adhered to coverslips within 24-well plates were fixed with 3.7% formaldehyde and stained with osmium tetroxide as described previously [16]. The morphology of the fixed cells was observed, and lipid bodies were counted by light microscopy with a 100x objective lens in 50 consecutively scanned macrophages. Resident peritoneal macrophage harvesting and treatments Western blotting analysis For in vitro assays, macrophages were obtained by peritoneal lavage with cold RPMI 1640. Then, cells were centrifuged at 4006 g for 10 minutes. Macrophages (36105/well) were cultured in 1 mL of RPMI 1640 medium supplemented with 1% Nutridoma-SP, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin in 24-well plates for 24 hours. Next, the macrophages were stimulated with different doses of L. longipalpis SGS (0.2, 0.5, 1.0, 1.5, 2.0 pairs/well). In some experiments, LPS (500 ng/well) was used as a positive control. One, 6, 24, 48 and 72 hours after stimuli, supernatants were collected and cells were fixed with 3.7% formaldehyde. For inhibitory assays, macrophages were pretreated for one hour with 1 mM NS-398, a COX-2 inhibitor; 20 gM BIS, a PKC inhibitor; or 50 mM PD98059, an ERK-1/2 inhibitor. Then, the cells were stimulated with SGS (1.5 pairs/well) or medium containing vehicle (DMSO) for 24 hours, and the supernatants were collected for eicosanoid measurement. Cell viability as assessed by trypan blue exclusion was always greater than 95% after the end of treatment. Macrophages were treated or not with SGS (1.0 pair/well) for 40 min. Next, the cells were washed once with phosphate-buffered saline, homogenized in lysis buffer containing phosphatase inhibitors (10 mM TRIS-HCl, pH 8.0, 150 mM NaCl, 0.5% v/ v Nonindet-P40, 10% v/v glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM sodium orthovanadate, 25 mM NaF and 1 mM PMSF) and a protease inhibitor cocktail (Roche, Indianapolis, IN). Protein concentrations were determined using the method of Lowry et al. [17] with BSA as the standard. Total proteins (20 mg) were then separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as described previously [18] and transferred onto nitrocellulose membranes. The membranes were blocked in Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TT) plus 5% BSA for 1 h before incubation overnight in the primary rabbit anti-mouse PKC-a and anti-ERK-1/2 (1:1,000) antibodies. After removal of the primary antibody and washing five times in TT, the membranes were incubated in the secondary antibody conjugated to peroxidase (1:10,000) for 1 h. Figure 1. Leukocyte influx into the peritoneal cavity of C57BL/6 mice in response to L. longipalpis SGS. Mice were injected i.p. with endotoxin-free saline or SGS (0.5 pair/cavity). One (A), 3 (B) and 6 (C) hours after stimulation, cells were harvested by peritoneal lavage and differential leukocyte counts were performed on Diff-quick stained cytospin preparations. The data are the means and SEM from an experiment representative of three independent experiments. Groups were compared using Student’s t test at each time point. *, p,0.05 and ***, p,0.001. doi:10.1371/journal.pntd.0000873.g001 www.plosntds.org 3 November 2010 | Volume 4 | Issue 11 | e873 36 Sand Fly SGS Triggers Eicosanoid Production Washed blots were then incubated with an ECL chemiluminescence kit (Amersham, UK). The membranes were discharged and immunoblotted again using primary rabbit anti-mouse phosphorylated-PKC-a and ERK-1/2 (1:1,000) antibodies according to the manufacturer’s instructions (Amersham, UK). Quantification of the level of proteins in the western blotting membranes was determined by densitometry. Briefly, bands were scanned and processed using Adobe Photoshop 5.0 software (Adobe Systems Inc.), and arbitrary values for protein density were estimated. Ratios between phosphorylated and unphosphorylated proteins were obtained to calculate the difference between groups. Results Lipid bodies and eicosanoids in leukocytes recruited by L. longipalpis SGS To measure the leukocyte recruitment induced by SGS, we injected 100 mL of saline or SGS (0.5 pair/cavity), and 1, 3 and 6 hours after injection, we enumerated total leukocytes recruited to the peritoneal cavity. Most of the cells recruited were mononuclear cells and neutrophils (Figure 1). In this context, SGS induced mononuclear cell recruitment for 3 hours (Figure 1 A and B) and neutrophil recruitment for over 6 hours (Figure 1A– C) of stimulation when compared with the saline group. Other cell populations (eosinophils and mast cells) were not altered after SGS stimulation, and there was no variation in these numbers over time (Figure 1). The peritoneal cell population in unstimulated animals (time zero) was composed of mononuclear cells (2.9856104 60.027) and negligible amounts of neutrophils (0.0186104 60.027). At this time, macrophages are the major cells within PGE2 and LTB4 measurement C57BL/6 mice were inoculated i.p. with 0.1 mL of L. longipalpis SGS (0.5 pair/cavity), endotoxin-free saline or 0.1 mL of LPS (500 gg/mL). At 1, 3 and 6 h post-stimulus, leukocytes were harvested by peritoneal washing with HBSS2/2 and 16106 cells/mL were resuspended in HBSS+/+ and stimulated with A23187 (0.5 mM) for 15 min [16]. The reactions were stopped on ice, and the samples were centrifuged at 5006 g for 10 min at 4uC. Supernatants from leukocytes re-stimulated ex vivo or those of in vitro assays were collected for measurement of PGE2 and LTB4 by enzyme-linked immunoassay (EIA) according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI). Statistical analysis The in vivo assays were performed using at least five mice per group. Each experiment was repeated at least three times. Data are reported as the mean and standard error of representative experiments and were analyzed using GraphPad Prism 5.0 software. Disparities in leukocyte recruitment, lipid bodies and lipid mediator quantification were explored using Student’s t test. Means from different groups from the in vitro assays were compared by ANOVA followed by Bonferroni’s test or a posttest for linear trends. Differences were considered statistically significant when p#0.05. Figure 3. Lipid body formation induced by SGS in vivo. C57BL/6 mice were injected i.p. with saline or SGS (0.5 pair/cavity). One, 3, 6 and 24 hours after stimulation, cells were harvested from the peritoneal cavity and stained with the neutral lipid probe BODIPY 493/503. Kinetics of LB formation in mononuclear (A) and polymorphonuclear (B) cells. Mean fluorescence intensity (MFI) histograms of mononuclear (C) and polymorphonuclear (D) cell populations at the 3-hour time point. Dotted lines indicate unstained cells, full lines indicate stained cells from the saline group (empty curves) and from the SGS-treated group (filled curves). LBs in mononuclear cells stimulated with saline (E) or SGS (F) for 3 h detected by fluorescence microscopy, nuclei stained with DAPI. Groups were compared using Student’s t test at each time point. *, p,0.05. MO, mononuclear; PMN, polymorphonuclear. doi:10.1371/journal.pntd.0000873.g003 Figure 2. Kinetics of eicosanoid production in response to L. longipalpis SGS ex vivo. C57BL/6 mice were injected i.p. with saline or SGS (0.5 pair/cavity). One, 3 and 6 hours after stimulation, peritoneal cavities were washed and cells were harvested. The cells were then incubated with A23187 (0.5 mM) for 15 min at 37uC to evaluate LTB4 and PGE2 production. The concentrations of PGE2 (A) and LTB4 (B) in the supernatant were measured by ELISA. The data are the means and SEM from an experiment representative of three independent experiments. Groups were compared using Student’s t test at each time point. *, p,0.05. doi:10.1371/journal.pntd.0000873.g002 www.plosntds.org 4 November 2010 | Volume 4 | Issue 11 | e873 37 Sand Fly SGS Triggers Eicosanoid Production Figure 4. Effect of L. longipalpis SGS on lipid body formation in peritoneal macrophages in vitro. Representative image of peritoneal macrophages untreated (A) or stimulated with SGS (1.5 pair/well) (B) for 24 hours. Dose-response (C) and kinetics (D) of lipid body formation induced by SGS in peritoneal macrophages. **, p,0.01 and ***, p,0.001 compared with unstimulated cells. doi:10.1371/journal.pntd.0000873.g004 Figure 5. COX-2 and PGE-synthase co-localize within lipid bodies induced by L. longipalpis SGS. Peritoneal macrophages were stimulated with SGS (1.5 pair/well) for 24 hours. BODIPY probe-labeled lipid bodies were visualized as green punctuate intra-cytoplasmic inclusions (A and D). COX-2 (B) and PGE-synthase (E) were localized with anti-COX-2 and anti- PGE-synthase antibodies, respectively. Merged images show co-localization of COX-2 (C) and PGE-synthase (F) within lipid bodies. doi:10.1371/journal.pntd.0000873.g005 www.plosntds.org 5 November 2010 | Volume 4 | Issue 11 | e873 38 Sand Fly SGS Triggers Eicosanoid Production the mononuclear population in the peritoneal cavity besides lymphocytes, which represent ,10% of mononuclear cells (data not shown). As shown in Figure 2, SGS administration led to enhanced PGE2 (Figure 2A) and LTB4 (Figure 2B) release within those cells recruited to the peritoneal cavity. Because LBs are sites of eicosanoid production [19], we evaluated LB formation in leukocytes recruited to the peritoneal cavity by FACs using the neutral lipid probe BODIPY 493/503. The kinetics of LB formation was evaluated at 1, 3, 6 and 24 hours after SGS stimulation by measuring mean fluorescence intensity (MFI). SGS increased MFI in mononuclear but not in polymorphonuclear cells after 3 and 6 hours, (Figure 3A and B) compared with the saline group. Histograms (Figure 3C and D) and fluorescence microscopic images (Figures 3E and F) at the 3-hour time point confirmed these effects of SGS on macrophages. L. longipalpis SGS triggers LB biogenesis in peritoneal macrophages in vitro To assess the role of SGS in lipid body formation in resident macrophages, we stimulated these cells with different doses of SGS (0.2–2.0 pairs/well) for different time periods (1, 6, 24, 48 and 72 hours). At 24 hours post-stimulus, SGS strongly induced LB formation compared with the untreated group (Figure 4A–D). LB formation was induced in a dose-dependent manner, and the maximum of LBs per macrophage was observed at a dose of 2.0 pairs/well (Figure 4C). Because LB formation induced by SGS (1.5 pairs/well) was more evident at 24 hours (Figure 4D), we selected this time point to perform further experiments. L. longipalpis SGS induces macrophage PGE2 production via the COX-2 enzyme Prostaglandins are produced by cyclooxygenases, which occur in constitutive (COX-1) and inducible (COX-2) forms [20]. We investigated the expression and subcellular localization of COX-2 within SGS-stimulated macrophages. Immunofluorescence microscopy revealed the presence of COX-2 (Figure 5A–C) and PGE-synthase (Figure 5D–F) within LBs in macrophages stimulated with SGS. Next, we measured PGE2 and LTB4 production in the supernatant of macrophage cultures. SGS induced PGE2 production starting at 1.0 pair/well (Figure 6A), whereas LTB4 was not detectable under any conditions (data not shown). As expected, PGE2 production by macrophages stimulated with SGS was reduced to basal levels when the cells were pre-incubated with NS398, a COX-2 inhibitor (Figure 6B). Thus, the PGE2 production in peritoneal macrophages induced by SGS occurs in newly formed lipid bodies and is dependent on COX-2. Figure 6. L. longipalpis SGS induces PGE2 production via COX-2. A, Dose-response of PGE2 production induced by SGS in peritoneal macrophages. B, Macrophages were pre-treated for 1 hour with the COX2 inhibitor N-398 before incubation with SGS (1.5 pair/well). Twenty-four hours after stimulation, PGE2 was measured in the supernatant. The data are the means and SEM from a representative experiment of three independent experiments. **, p,0.01 and #, p,0.05. doi:10.1371/journal.pntd.0000873.g006 Discussion Sand fly saliva triggers an inflammatory response characterized by cellular influx followed by hemostatic and immune mechanism suppression. Nevertheless, the role of sand fly saliva in eicosanoid production during the early steps of the innate immune response is poorly understood. In inflammatory conditions, eicosanoids are mostly produced in cytoplasmic organelles called lipid bodies (LBs), which are formed in leukocytes and other cells involved in the inflammatory and infectious responses to several stimuli [13]. Herein, we showed that L. longipalpis saliva induces lipid body formation and PGE2 production in peritoneal macrophages ex vivo and in vitro via kinase phosphorylation and COX-2 activation. Previous investigations have demonstrated that sand fly saliva plays an important role in cellular recruitment in multiple experimental models [3,9,11,12], including in vivo sand fly bites [22]. Herein, we confirmed previous reports that L. longipalpis SGS induces an inflammatory infiltration composed mainly of macrophages and neutrophils. Moreover, we showed that the cellular recruitment induced by L. longipalpis saliva is concomitant with PGE2 and LTB4 production. In this scenario, lipid mediators SGS induces PGE2 production via PKC-a and ERK-1/2 Multiple pathways are involved in the signaling for PGE2 production [13]. Recently, ERK and PKC-a were shown to be involved in COX-2 activity [21]. We observed that SGS activated both ERK (Figure 7A and C) and PKC-a phosphorylation (Figure 7B and D), but it did not alter the levels of the unphosphorylated proteins. To investigate whether these kinases are involved in the induction of PGE2 production by SGS, we pretreated macrophages with bisindolylmaleimide I (BIS I) and PD98059, PKC-a and ERK-1/2 inhibitors, respectively (Figure 8A–B). Inhibition of both enzymes completely abrogated PGE2 production induced by SGS (Figure 8A–B). In sum, these results suggest that PKC-a and ERK-1/2 are involved in the PGE2 production induced by SGS. www.plosntds.org 6 November 2010 | Volume 4 | Issue 11 | e873 39 Sand Fly SGS Triggers Eicosanoid Production Figure 7. L. longipalpis SGS induces PKC-a and ERK phosphorylation. Peritoneal macrophages were incubated in the absence (control) or presence of SGS (1.5 pair/mL) for 40 min. The cells were lysed and immunoblotted using polyclonal anti-ERK-1/2 (A) or anti-PKCa (B) antibodies. The membranes was discharged and immunoblotted using polyclonal anti- phospho-ERK-1/2 (A) or anti- phosphor-PKC-a (B) antibodies. Quantification of phosphorylated-ERK-1/2 (C) and phosphorylated-PKCa (D) was determined by densitometry. The data show the fold increase in the phosphorylated/unphosphorylated kinase ratio of the SGS group relative to the control group. P-, phosphorylated. doi:10.1371/journal.pntd.0000873.g007 could be triggering cellular recruitment. Secretion of LTB4 by resident macrophages plays an important role in neutrophil migration [23]. In addition, lipopolysaccharides induce macrophage migration via prostaglandin D2 and prostaglandin E2 [10]. Prostaglandin E2 is an abundant eicosanoid produced by inflammatory cells, and it is known to exert anti-inflammatory and vasodilator effects. PGE2 is found in Ixodes scapularis saliva and is also implicated in the immunomodulatory activity of tick saliva on dendritic cell and macrophage activation [24]. Furthermore, previous studies using saliva from several Phlebotomus species have suggested that the anti-inflammatory properties of sand fly saliva could be attributed to PGE2 and IL-10 released by dendritic cells [9,25]. In these studies, the cellular recruitment induced by OVA stimulation was abrogated by saliva from various sand fly species [9,25], which was associated with an anti-inflammatory profile dependent on the production of IL-10, IL-4 [25] and PGE2 [9]. Intriguingly, maxadilan, a vasodilator peptide with immunomodulatory activities present in L. longipalpis saliva, is able to induce LPS-activated macrophages to release PGE2 via COX-1, an enzyme that is constitutively active [7]. In the present study, we showed that L. longipalpis SGS triggers PGE2 production in resident macrophages by an inducible pathway, since this effect was completely abrogated when the cells were incubated in the presence of NS-398, a COX-2 inhibitor. Nevertheless, whether sand fly saliva contains other molecules involved in PGE2 production or pharmacological amounts of this mediator similarly to tick saliva remains unknown. Our study is the first to establish a direct link between L. longipalpis saliva, eicosanoid production and lipid body formation. Under inflammatory and infectious conditions, lipid mediators are mainly produced within LBs, which compartmentalize both the substrate and the enzymatic machinery required for eicosanoid production [13]. In this regard, the enzymes COX and 5-LO have www.plosntds.org Figure 8. ERK and PKC kinase inhibitors abrogate PGE2 production induced by L. longipalpis SGS. Peritoneal macrophages were pre-treated for 1 hour with BIS I (A) or PD98059 (B) before incubation with SGS (1.5 pair/well). Twenty-four hours after stimulation, PGE2 was measured in the supernatant. The data are the mean and SEM from an experiment representative of three independent experiments. ***, p,0.001; ##, p,0.01 and ###, p,0.001. PD98059, ERK inhibitor; BIS-I, PKC inhibitor. doi:10.1371/journal.pntd.0000873.g008 been localized to lipid bodies in various inflammatory cells by the use of multiple techniques including fluorescence microscopy [13]. Previous studies have shown that various inflammatory and infectious stimuli are able to trigger LB formation in macrophages [13,19]. Our findings demonstrate that SGS induces LB formation in macrophages in vivo and in vitro, suggesting that L. longipalpis saliva acts directly on these cells, but not on neutrophils. Indeed, L. longipalpis SGS triggered LB formation in macrophages committed to PGE2 production via COX-2 and PGE-synthase. Data regarding the direct effects of sand fly salivary compounds on host signaling pathways cells are scarce. The extracellular signal-regulated kinases (ERKs) and protein kinase C (PKC) are among the key enzymes implicated in signaling pathways of diverse cellular responses, including eicosanoid production. The MAP kinases ERK1 and ERK2 induce activation of cPLA2, an enzyme that hydrolyzes arachidonic acid, which is metabolized to 7 November 2010 | Volume 4 | Issue 11 | e873 Sand Fly SGS Triggers Eicosanoid Production prostaglandin H2 by COX [13]. Previous studies have demonstrated the compartmentalization of MAP kinases and cPLA2 at arachidonate-enriched lipid bodies [26,27], as well as COX-2 and PGE-synthase [16,28,29]. Herein, it is shown for the first time that L. longipalpis SGS triggers ERK-1/2 and PKC-a phosphorylation in macrophages. Other studies have shown that COX-2 activation and PGE2 production in LPS stimulated-macrophages is dependent on the phosphorylation of protein kinases such as PKC-a [21] and ERK-1/2 [30]. We showed that the PGE2 production induced by SGS is dependent on both ERK-1/2 and PKC. This association between the activation of kinases and the metabolism of eicosanoids within lipid bodies may serve to enhance rapid eicosanoid production in response to extracellular stimuli such as sand fly saliva. Of note, in addition to their role in regulating the host response to infection by modulating inflammatory mediator production, lipid bodies may also serve as rich sources of nutrients for intracellular pathogens, thus favoring intracellular pathogen replication [31,32]. In brief, the present work provides new insights into the mechanisms involved in macrophage responses to L. longipalpis saliva, including LB formation and the signaling pathways that trigger PGE2 release. Although the roles of the newly formed LBs and PGE2 induced by sand fly saliva in the pathogenesis of leishmaniasis have not yet been addressed, several studies have shown that PGE2 is essential to the infection of macrophages [33,34] and parasite dissemination after infection [35]. The induction of PGE2 production by sand fly saliva demonstrated herein can influence the initial steps of host infection by favoring less intense macrophage activation. Our group and others have been providing strong evidence that saliva components are immunogenic and have potential as markers of exposure to sand fly vectors [36–39]. Further studies are required to determinate if the immunization based on components of vector saliva interferes in eicosanoid production with consequences for the host’s immune response and the transmissibility of the parasite. Acknowledgments We thank Dr. Manoel Barral-Netto for critical discussion of the manuscript. We also gratefully acknowledge the technical assistance of Edvaldo Passos, Marcos Fonseca and to Dr. Clarissa M. Maya-Monteiro and Dr. Heloiza D’Ávila for intellectual contributions. Author Contributions Conceived and designed the experiments: TAS DBP BBA DON JC PFE CIB AB PTB VMB. Performed the experiments: TAS DBP BBA DON JC PFE. Analyzed the data: TAS DBP BBA DON JC PFE CIB PTB VMB. Contributed reagents/materials/analysis tools: ABC MACSN JCM PTB VMB. Wrote the paper: TAS DBP BBA PTB VMB. References 16. Pacheco P, Bozza FA, Gomes RN, Bozza M, Weller PF, et al. (2002) Lipopolysaccharide-induced leukocyte lipid body formation in vivo: innate immunity elicited intracellular Loci involved in eicosanoid metabolism. J Immunol 169: 6498–6506. 17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275. 18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. 19. Bozza PT, Melo RC, Bandeira-Melo C (2007) Leukocyte lipid bodies regulation and function: Contribution to allergy and host defense. Pharmacol Ther 113: 30–49. 20. Brock TG, Peters-Golden M (2007) Activation and regulation of cellular eicosanoid biosynthesis. ScientificWorldJournal 7: 1273–1284. 21. Giroux M, Descoteaux A (2000) Cyclooxygenase-2 expression in macrophages: modulation by protein kinase C-alpha. J Immunol 165: 3985–3991. 22. Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, et al. (2008) In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321: 970–974. 23. Oliveira SH, Canetti C, Ribeiro RA, Cunha FQ (2008) Neutrophil migration induced by IL-1beta depends upon LTB4 released by macrophages and upon TNF-alpha and IL-1beta released by mast cells. Inflammation 31: 36–46. 24. Sa-Nunes A, Bafica A, Lucas DA, Conrads TP, Veenstra TD, et al. (2007) Prostaglandin E2 is a major inhibitor of dendritic cell maturation and function in Ixodes scapularis saliva. J Immunol 179: 1497–1505. 25. Monteiro MC, Nogueira LG, Almeida Souza AA, Ribeiro JM, Silva JS, et al. (2005) Effect of salivary gland extract of Leishmania vector, Lutzomyia longipalpis, on leukocyte migration in OVA-induced immune peritonitis. Eur J Immunol 35: 2424–2433. 26. Yu W, Bozza PT, Tzizik DM, Gray JP, Cassara J, et al. (1998) Cocompartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies. Am J Pathol 152: 759–769. 27. Moreira LS, Piva B, Gentile LB, Mesquita-Santos FP, D’Avila H, et al. (2009) Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acidsinduced lipid bodies of epithelial cells. Biochim Biophys Acta 1791: 156–165. 28. D’Avila H, Melo RC, Parreira GG, Werneck-Barroso E, Castro-Faria-Neto HC, et al. (2006) Mycobacterium bovis bacillus Calmette-Guerin induces TLR2mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo. J Immunol 176: 3087–3097. 29. Accioly MT, Pacheco P, Maya-Monteiro CM, Carrossini N, Robbs BK, et al. (2008) Lipid bodies are reservoirs of cyclooxygenase-2 and sites of prostaglandinE2 synthesis in colon cancer cells. Cancer Res 68: 1732–1740. 30. West MA, Clair L, Bellingham J, Wahlstrom K, Rodriguez JL (2000) Defective lipopolysaccharide-dependent ERK 1/2 activation in endotoxin tolerant murine macrophages is reversed by direct protein kinase C stimulation. Shock 14: 169–175. 31. D’Avila H, Maya-Monteiro CM, Bozza PT (2008) Lipid bodies in innate immune response to bacterial and parasite infections. Int Immunopharmacol 8: 1308–1315. 1. Andrade BB, Teixeira CR, Barral A, Barral-Netto M (2005) Haematophagous arthropod saliva and host defense system: a tale of tear and blood. An Acad Bras Cienc 77: 665–693. 2. Peters NC, Sacks DL (2009) The impact of vector-mediated neutrophil recruitment on cutaneous leishmaniasis. Cell Microbiol 11: 1290–1296. 3. Silva F, Gomes R, Prates D, Miranda JC, Andrade B, et al. (2005) Inflammatory cell infiltration and high antibody production in BALB/c mice caused by natural exposure to Lutzomyia longipalpis bites. Am J Trop Med Hyg 72: 94–98. 4. Vinhas V, Andrade BB, Paes F, Bomura A, Clarencio J, et al. (2007) Human anti-saliva immune response following experimental exposure to the visceral leishmaniasis vector, Lutzomyia longipalpis. Eur J Immunol 37: 3111–3121. 5. Costa DJ, Favali C, Clarencio J, Afonso L, Conceicao V, et al. (2004) Lutzomyia longipalpis salivary gland homogenate impairs cytokine production and costimulatory molecule expression on human monocytes and dendritic cells. Infect Immun 72: 1298–1305. 6. Zer R, Yaroslavski I, Rosen L, Warburg A (2001) Effect of sand fly saliva on Leishmania uptake by murine macrophages. Int J Parasitol 31: 810–814. 7. Soares MB, Titus RG, Shoemaker CB, David JR, Bozza M (1998) The vasoactive peptide maxadilan from sand fly saliva inhibits TNF-alpha and induces IL-6 by mouse macrophages through interaction with the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor. J Immunol 160: 1811–1816. 8. Wilborn J, DeWitt DL, Peters-Golden M (1995) Expression and role of cyclooxygenase isoforms in alveolar and peritoneal macrophages. Am J Physiol 268: L294–301. 9. Carregaro V, Valenzuela JG, Cunha TM, Verri WA, Jr., Grespan R, et al. (2008) Phlebotomine salivas inhibit immune inflammation-induced neutrophil migration via an autocrine DC-derived PGE2/IL-10 sequential pathway. J Leukoc Biol 84: 104–114. 10. Tajima T, Murata T, Aritake K, Urade Y, Hirai H, et al. (2008) Lipopolysaccharide induces macrophage migration via prostaglandin D(2) and prostaglandin E(2). J Pharmacol Exp Ther 326: 493–501. 11. Monteiro MC, Lima HC, Souza AA, Titus RG, Romao PR, et al. (2007) Effect of Lutzomyia longipalpis salivary gland extracts on leukocyte migration induced by Leishmania major. Am J Trop Med Hyg 76: 88–94. 12. Teixeira CR, Teixeira MJ, Gomes RB, Santos CS, Andrade BB, et al. (2005) Saliva from Lutzomyia longipalpis induces CC chemokine ligand 2/monocyte chemoattractant protein-1 expression and macrophage recruitment. J Immunol 175: 8346–8353. 13. Bozza PT, Magalhaes KG, Weller PF (2009) Leukocyte lipid bodies - Biogenesis and functions in inflammation. Biochim Biophys Acta 1791: 540–551. 14. Prates DB, Santos LD, Miranda JC, Souza AP, Palma MS, et al. (2008) Changes in amounts of total salivary gland proteins of Lutzomyia longipallpis (Diptera: Psychodidae) according to age and diet. J Med Entomol 45: 409–413. 15. Plotkowski MC, Brandao BA, de Assis MC, Feliciano LF, Raymond B, et al. (2008) Lipid body mobilization in the ExoU-induced release of inflammatory mediators by airway epithelial cells. Microb Pathog 45: 30–37. www.plosntds.org 8 November 2010 | Volume 4 | Issue 11 | e873 40 41 Sand Fly SGS Triggers Eicosanoid Production 32. Bozza PT, D’Avila H, Almeida PE, Magalhães KG, Molinaro R, et al. (2009) Lipid droplets in host–pathogen interactions. Clinical Lipidology 4: 791–807. 33. Afonso L, Borges VM, Cruz H, Ribeiro-Gomes FL, DosReis GA, et al. (2008) Interactions with apoptotic but not with necrotic neutrophils increase parasite burden in human macrophages infected with Leishmania amazonensis. J Leukoc Biol 84: 389–396. 34. Matte C, Maion G, Mourad W, Olivier M (2001) Leishmania donovani-induced macrophages cyclooxygenase-2 and prostaglandin E2 synthesis. Parasite Immunol 23: 177–184. 35. Anstead GM, Chandrasekar B, Zhao W, Yang J, Perez LE, et al. (2001) Malnutrition alters the innate immune response and increases early visceralization following Leishmania donovani infection. Infect Immun 69: 4709–4718. www.plosntds.org 36. Barral A, Honda E, Caldas A, Costa J, Vinhas V, et al. (2000) Human immune response to sand fly salivary gland antigens: a useful epidemiological marker? Am J Trop Med Hyg 62: 740–745. 37. Gomes RB, Brodskyn C, de Oliveira CI, Costa J, Miranda JC, et al. (2002) Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity. J Infect Dis 186: 1530–1534. 38. Souza AP, Andrade BB, Aquino D, Entringer P, Miranda JC, et al. Using recombinant proteins from Lutzomyia longipalpis saliva to estimate human vector exposure in visceral Leishmaniasis endemic areas. PLoS Negl Trop Dis 4: e649. 39. Teixeira C, Gomes R, Collin N, Reynoso D, Jochim R, et al. Discovery of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi in Latin America. PLoS Negl Trop Dis 4: e638. 9 November 2010 | Volume 4 | Issue 11 | e873 43 4.2. MANUSCRITO II New Insights on the Inflammatory Role of Lutzomyia longipalpis Saliva in Leishmaniasis Novas Ideias Sobre ao Papel Inflamatório da Saliva de Lutzomyia longipalpis na Leishmaniose Este trabalho revisa os principais achados do nosso grupo sobre o papel da saliva na resposta inflamatória durante os momentos iniciais da infecção por Leishmania. Nesta revisão destacamos o efeito da saliva sobre macrófagos e neutrófilos no que tange a modulação da produção de PGE2. A seção 4.1 intitulada “Eventos Inflamatórios Disparados pela Saliva de L. longipalpis” aborda dados preliminares que serão melhor discutidos no Manuscrito III desta tese. Na seção 5 intitulada “Resposta do Macrófago Hospedeiro à Saliva de L. longipalpis” encontramos um breve resumo dos dados apresentados no Manuscrito I desta tese, bem como uma discussão sobre os achados da literatura acerca do efeito da saliva sobre macrófagos. Por fim, na seção 6 intitulada “Neutrófilos e Saliva de L. longipalpis: Uma Interação Negligenciada sobre o Cenário da Infecção por Leishmania”, nós abordamos nossos achados sobre o efeito da saliva na indução da apoptose de neutrófilos murinos e humanos. Os dados desta última seção são apresentados em uma publicação de minha co-autoria intitulada “Lutzomyia longipalpis saliva drives apoptosis and enhances parasite burden in neutrophils”, a qual pode ser encontrada na seção Apêndice. Este artigo foi publicado no periódico internacional Journal of Parasitology Research 44 Hindawi Publishing Corporation Journal of Parasitology Research Volume 2012, Article ID 643029, 11 pages doi:10.1155/2012/643029 Review Article New Insights on the Inflammatory Role of Lutzomyia longipalpis Saliva in Leishmaniasis Deboraci Brito Prates,1, 2 Théo Araújo-Santos,2, 3 Cláudia Brodskyn,2, 3, 4 Manoel Barral-Netto,2, 3, 4 Aldina Barral,2, 3, 4 and Valéria Matos Borges2, 3, 4 1 Departamento de Biomorfologia, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Avenida Reitor Miguel Calmon S/N, 40110-100 Salvador, BA, Brazil 2 Centro de Pesquisa Gonçalo Moniz (CPqGM), Fundação Oswaldo Cruz (FIOCRUZ), Rua Waldemar Falcão 121, 40296-710 Salvador, BA, Brazil 3 Faculdade de Medicina da Bahia, Universidade Federal da Bahia, Avenida Reitor Miguel Calmon S/N, 40110-100 Salvador, BA, Brazil 4 Instituto Nacional de Ciência e Tecnologia de Investigação em Imunologia (iii-INCT), Avenida Dr.Enéas de Carvalho Aguiar 44, 05403-900, São Paulo, SP, Brazil Correspondence should be addressed to Valéria Matos Borges, [email protected] Received 15 August 2011; Revised 24 October 2011; Accepted 27 October 2011 Academic Editor: Marcela F. Lopes Copyright © 2012 Deboraci Brito Prates et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. When an haematophagous sand fly vector insect bites a vertebrate host, it introduces its mouthparts into the skin and lacerates blood vessels, forming a hemorrhagic pool which constitutes an intricate environment of cell interactions. In this scenario, the initial performance of host, parasite, and vector “authors” will heavily influence the course of Leishmania infection. Recent advances in vector-parasite-host interaction have elucidated “co-authors” and “new roles” not yet described. We review here the stimulatory role of Lutzomyia longipalpis saliva leading to inflammation and try to connect them in an early context of Leishmania infection. 1. Introduction Leishmaniasis remains a serious problem in public health, endemic in 88 countries on four continents, but most of the cases occur in underdeveloped or developing countries [1]. Visceral Leishmaniasis (VL) is a progressive infection with fatal outcome in the absence of treatment. Approximately 90% of the VL cases registered in the Americas occur in Brazil and are concentrated in the Northeast region. In the New World, Lutzomyia longipalpis is the principal vector of Leishmania infantum chagasi, the agent of American Visceral Leishmaniasis [2]. The causes related to development of distinct clinical manifestations in leishmaniasis are multifactorial and reflect the complexity at the vector-pathogen-host interface [3]. Protozoan parasites of the genus Leishmania are the causative agents of the disease and are transmitted to the mammalian hosts by the bite of female phlebotomine sand flies during blood repast. For blood meal obtainment, sand flies introduce their mouthparts into the skin, tearing tissues, lacerating capillaries, and creating haemorrhagic pools upon which they feed [4]. The presence of sand fly saliva in the blood pool, the environment where the parasite encounters host cells, influences the development and functions of several leukocytes. In recent years, the importance of the interaction between components of sand fly saliva and host immune mechanisms in regulating infectivity and disease progression has become clearer and suggests their consequences to disease outcome in leishmaniasis [5]. The aspects involved in immune response resulting in resistance or susceptibility widely depend on the first attempt of host’s innate response to contain infection that may influence on the predominance of a pattern of future host’s immune adaptive response against Leishmania. Many 45 2 studies have been performed to understand the mechanisms leading to protection or exacerbation of the disease however; relatively few studies have investigated the role of the sandfly-derived salivary compounds in the innate immunity. In this paper we integrate the influence of sand fly bite with current ideas regarding the role of early steps of host inflammatory response against Leishmania. 2. Sand Fly Saliva: A Rich Field of Study Sand fly vectors display a rich source of salivary biological active components to acquire blood from vertebrate hosts, a task not easy due the haemostatic, inflammatory and immune responses resultant from the bite [6]. Thus, it is not unexpected that many scientists have progressively investigated several aspects of sand fly saliva, concerning its composition and the range of mammalian response to it. Among the New World species of sand fly which are vectors of Leishmania, L. longipalpis and its salivary gland content are the best studied. One of the first components related to L. longipalpis salivary gland was maxadilan [7], the most potent vasodilator peptide known and one of the two phlebotomine salivary proteins more extensively studied. Maxadilan is recognized by causing typical erythema during the feeding of L. longipalpis [8]. Further, it was described that maxadilan is able to modulate the inflammatory response by inhibiting cytokines such as TNF-α, by inducing IL-6 production, and by stimulating hematopoiesis [9–11]. Charlab et al. (1999) reported nine full clones and two partial cDNA clones from salivary gland from L. longipalpis [12]. In that work, they reported for the first time a hyaluronidase activity from sand fly saliva, an activity not yet described on phlebotomine sand flies, helping the diffusion of other pharmacological substances through the skin matrix [13]. It was also described an apyrase activity on L. longipalpis saliva which hydrolyses ATP and ADP to AMP, functioning as a potent antiplatelet factor [12, 14]. Interestingly, a 5 nucleotidase activity is also present in L. longipalpis saliva exert vasodilator and antiplatelet aggregation role by converting AMP to adenosine [12]. One of the most abundant protein found in the L. longipalpis saliva is the Yellow-related protein [12, 13, 15, 16]. Our group has demonstrated that this family of proteins are the most recognized in sera from children living in an endemic area of visceral Leishmaniasis in Brazil [17] and by normal volunteers exposed to laboratory-reared L. longipalpis bites [18]. Recently, Xu et al. (2011) described the structure and function of a yellow pro-tein LJM 11 [19]. In this report, the authors described that yellow proteins from L. longipalpis saliva act as binder of proinflammatory biogenic amines such as serotonin, histamine, and catecholamines [19]. One member of the D7 family of proteins (commonly found in dipterans saliva) is present in L. longipalpis [12]. The exact function of this protein in sand fly saliva is still unknown. However, its role on mosquito’s saliva suggests that it could act as anticoagulant or binding biogenic amines avoiding host inflammatory events [12, 15]. Herein, we present some of the most studied proteins related to L. longipalpis saliva. (See [6, 15, 16, 20] for more Journal of Parasitology Research details about this topic). Although many of them have been associated with blood-feeding, their biological functions remain undefined. Nevertheless, by modulating the host haemostatic and inflammatory response, this yet unreported sand fly salivary content remains as a research challenge, acting on host immunity to Leishmania during transmission and establishment of infection. 3. Immune Response to Lutzomyia longipalpis Saliva against Leishmania There are several studies contributing to a better understanding of L. longipalpis saliva effects on host immunity to Leishmania infection. A brief exposition of these major contributions in the last 10 years is shown in Figure 1. In mice, salivary products seem to exacerbate the infection with Leishmania and may, in fact, be mandatory for establishment of the parasite in vertebrate hosts. It has been shown that components of L. longipalpis or Phlebotomus papatasi salivary gland lysates mixed with Leishmania major resulted in substantially larger lesions compared to controls [21, 22]. Our group have shown that repeated exposure of BALB/c mice to L. longipalpis bites leads to local inflammatory cell infiltration comprised of neutrophils, macrophages and eosinophils [23]. Total IgG and IgG1 antibodies react predominantly with three major protein bands (45, 44, and 16 kD) from insect saliva by Western blot [23]. The injection of immune serum previously incubated with salivary gland homogenate induced an early infiltration with neutrophils and macrophages, suggesting the participation of immune complexes in triggering inflammation [23]. We have shown that in endemic areas natural exposures to noninfected sand fly bites can influence the epidemiology of the disease [17, 24]. We observed that people who presented antibodies against saliva of L. longipalpis also showed DTH anti-Leishmania, suggesting that the immune response against saliva of the vector could contribute to the induction of a protective immune response against the parasite. Recently, in a prospective study this data was reinforced by Aquino et al. (2010) evaluating 1,080 children from 2 endemic areas for VL [25]. There was a simultaneous appearance of antibodies anti-saliva and an anti-Leishmania DTH, or a cellular response against the parasite [25], sup-porting the idea that eliciting immunity against saliva could benefit the induction of a protective response against the parasite. The anti-sand fly antibodies can serve as epidemiological marker of vector exposure in endemic areas. In fact, we demonstrated that two salivary proteins, called LJM 17 and LJM 11, were specifically recognized by humans exposed to L. longipalpis, but not Lutzomyia intermedia [26]. We also evaluated the specificity of anti-L. longipalpis in a panel of 1,077 serum samples and verified that LJM 17 and LJM 11 together in an ELISA assay identified the effectiveness of these proteins for the prediction of positivity against salivary gland sonicate (SGS) [27]. In experimental model using C57BL/6 mice, immunization with LJM 11 triggered DTH response and decrease the diseased burden after L. major infection [19]. 46 Journal of Parasitology Research 3 Sand fly Skin Saliva Resident macrophage Chemotaxis Silva et al. 2005 [23] Teixeira et al. 2005 [42] Araújo-Santos et al., 2010 [51] ↓CD80 ↑HLA-DR Costa et al., 2004 [28] ↑PGE2 via PKC-α and ERK-1/2 ↑CD4+ CD25+ ↑CD8+ CD25+ Vinhas et al., 2007 [18] Araújo-Santos et al., 2010 [51] Red blood cell ↓IL-10 and TNF-α ↑IL-6, IL-8 and IL-12 p40 ↑CD86 and HLA-DR Dendritic cell Costa et al., 2004 [28] ↑IgG, IgG4 and IgG1 Barral et al., 2000 [17] Gomes et al., 2002 [24] Silva et al., 2005 [23] Vinhas et al., 2007 [18] Macrophage Monocyte ↑Parasite burden ↑FasL and apoptosis ↑MCP-1, PGE2 ↓ROS Eosinophil Neutrophil T lymphocyte B lymphocyte ↓ CD80, CD86 and HLA-DR Prates et al., 2011 [76] Costa et al., 2004 [28] Figure 1: Roles of Lutzomyia longipalpis saliva in host immune response cell. After L. longipalpis saliva injection a set of events can be triggered in the host immune response. Herein, we summarized the roles of saliva on major cell populations involved in the host immune response against Leishmania infection. We also characterized the immunological patterns following sand fly saliva exposure, using healthy volunteers exposed to laboratory-reared L. longipalpis [18]. We noticed high levels of IgG1, IgG4, and IgE antibodies antisaliva. Furthermore, following in vitro stimulation with salivary gland sonicate, there was an increased frequency of CD4(+)CD25(+) and CD8(+)CD25(+) T cells as well as IFN-γ and IL-10 synthesis. Strikingly, 1 year after the first exposure, PBMC from the volunteers displayed recall IFNγ responses that correlated with a significant reduction in infection rates using a macrophage-lymphocyte autologous culture. Together, these data suggest that human immunization against sand fly saliva is feasible and recall responses are obtained even 1 year after exposure, opening perspectives for vaccination in man [18]. Sand fly saliva also seems to exert a direct effect on human antigen presenting cells. L. longipalpis SGS inhibited IL-10 and TNF-α production but induced IL-6, IL-8, and IL-12p40 production by LPS-stimulated monocytes and dendritic cells [28]. Besides cytokine production, sand fly saliva also interfered with the expression of costimulatory molecules in macrophages (reduced CD80 and increased HLA-DR expression) and in monocytes (increased CD80 and HLA-DR expression). During dendritic cell differentiation induced by CD40L, a slight reduction in CD80, CD86, HLADR, and CD1a expression were also observed [28]. Whereas enhancement of Leishmania transmission by saliva is probably due to immunomodulatory components of sand fly saliva, an explanation of the anti-Leishmania effect resulting from host immunization against salivary antigen is not straightforward. Immunity in this system could derive from neutralization of salivary immunomodulators such as the peptide maxadilan from L. longipalpis (as reviewed in [22]). Alternatively, immunity could derive from a DTH reaction at the site of the bite generated by a cellular response to salivary antigens injected by the fly [29, 30]. This particular reaction could turn the lesion and its surroundings into an inhospitable site for the establishment of Leishmania infection in the new host, or it could modify the environment priming the initial events of the host immune reaction to Leishmania. The disease exacerbative properties of saliva, often resulting from the bioactive property of one or more of its molecules, should not be confounded with antigenic molecules in saliva that induce an adaptive immune response in the host. This acquired immunity can be either protective 47 4 or exacerbative depending on the nature and dominance of the salivary components of a vector species. Exposure to uninfected bites of the sand fly P. papatasi induces a strong delayed-type hypersensitivity response and IFN-γ production at the bite site that confers protection in mice challenged by L. major-infected flies [29]. By contrast, acquired immunity to L. intermedia saliva results in disease exacerbation not protection [31]. Moreover, P. papatasi saliva, despite its overall protective property, contains molecules that alone induce a protective (PpSP15) or exacerbative (PpSP44) im-mune response in the host [32, 33]. It is likely that L. intermedia saliva also contains molecules with similar profiles despite the overall exacerbative effect of total saliva. Recently, we developed a model for visceral Leishmaniasis (VL) in hamsters, using an intradermal inoculation in the ears of 100,000 L. chagasi parasites together with L. longipalpis saliva to mimic natural transmission by sand flies [34]. Hamsters developed classical signs of VL rapidly, culminating in a fatal outcome 5-6 months postinfection. Immunization with 16 DNA plasmids coding for salivary proteins of L. longipalpis resulted in the identification of LJM19, a novel 11-kDa protein that protected hamsters against the fatal outcome of VL. LJM19-immunized hamsters maintained a low parasite load that correlated with an overall high IFN-γ/TGF-β ratio and inducible NOS expression in the spleen and liver up to 5 months post-infection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-γ was also noted in the skin of LJM19immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-γ at the site of bite could partly explain the protection observed in the viscera of LJM19immunized hamsters through direct parasite killing and/or priming of anti-Leishmania immunity. Recently, Tavares et al. [35] showed that LJM19 was also able to protect hamsters against an infection composed by Leishmania braziliensis plus saliva of L. intermedia, the vector responsible for the transmission of this parasite in Brazil [35]. The immunization also induced a higher ratio of IFN-γ/TGFβ production in the cells from lymph nodes draining the infection site. Collin et al., (2009) immunized dogs using intradermal injections of DNA codifying salivary proteins of L. longipalpis (LJM17 and LJL 143), followed by injection of recombinant Canarypox virus containing the same genes [36]. They also observed a potential protective response against Leishmania, showing high concentrations of IFNγ in PBMC stimulated with recombinant salivary proteins. Importantly, the bite of uninfected sand flies resulted in a strong DTH characterized by high amount of IFN-γ and low levels of TGF-β [36]. Together, these results point out the possibility to immunize against leishmaniasis using defined proteins of vector’s saliva against Leishmania. 4. Early Steps of Host-Vector-Leishmania Interplay: Cell Recruitment Induced by Saliva It is well established that the first steps in leishmaniasis are critical in determining the development of the disease. In order to understand this critical moment, several reports Journal of Parasitology Research have investigated the early recruitment of cells induced by both L. longipalpis saliva alone or coinoculated with L. chagasi. Sand fly saliva is able to induce an inflammatory process in the host by recruiting different cells into the bite site. In fact, it was verified that L. longipalpis salivary gland lysate markedly modifies the inflammatory response to infection with L. braziliensis in BALB/c mice [37]. The salivaassociated lesions progressed to extensive accumulations of heavily parasitized epithelioid macrophages, with persistent neutrophilia and eosinophilia [37]. Eosinophilia has also been described in dogs intradermally inoculated with L. longipalpis saliva associated with L. chagasi promastigotes [38]. Interestingly, this inflammatory response was not observed in animals that received saliva or parasites alone [38]. The significance of this in the context of Leishmaniasis remains to be investigated. However, this phenomena is not exclusive to L. longipalpis saliva once eosinophils were described in the inflammatory course at the site of immunization of mice with the salivary recombinant 15kDa protein from P. papatasi, the sand fly species vector of Leishmania major [32]. It is well established the abundant presence of eosinophils in both inflammatory site and allergic response. Activated eosinophils release lipid mediators as PAF, prostaglandins, leukotrienes, and lipoxins, as well as cytokines IL-10 and IL-8 that, in conjunct, trigger vasodilatation and leukocyte chemotaxis (reviewed in [39]). In the context of sand fly bite, this eosinophilic reaction could favor vector feeding but creates an unfriendly environment for Leishmania parasites. Host cell infiltration induced by sand fly bite is the most physiologic approach to reinforce the inflammatory role of vector saliva. This event has been explored using P. papatasi, in which saliva-induced DTH response observed was associated to a possible fly adaptation to manipulate host immunity for the vector’s own advantage [30]. Concerning L. longipalpis saliva, our group investigated the initial vertebrate reactions against sand fly saliva. We demonstrated that repeated exposures of BALB/c mice to L. longipalpis bites lead to an intense and diffuse inflammatory infiltrate characterized by neutrophils, eosinophils, and macrophages [23]. This response was observed by histological analysis of the ear dermis from exposed mice as early as 2 hours and was sustained up to 48 hours after challenge with the L. longipalpis salivary sonicate [23]. Moreover, the injection of immune serum previously incubated with salivary gland homogenate induced an early infiltration with neutrophils and macrophages, suggesting the participation of immune complexes in triggering inflammation [23]. An elegant and remarkable visual advance obtained by two-photon intravital imaging has recently demonstrated that the neutrophils represent the first cell population which is recruited to Phlebotomus duboscqi bite site [40]. Although the participation of vector salivary components had not been directly attributed to this inflammatory event by the authors, we could not discharge this possibility considering diverse data showing that saliva from different sand flies species exert chemotaxis. As neutrophils were observed on L. longipalpis bite site [23] the implications of its saliva on this cells will be further discussed in this paper. 48 Journal of Parasitology Research In addition to in vivo models, cell chemotaxis induced by saliva has also been observed in vitro. This is of particular interest, indicating that L. longipalpis salivary components can act directly as inflammatory mediator. Using transwell system, Zer et al. (2001) showed the direct chemotatic effect of saliva on BALB/c peritoneal macrophages. In the same work, it was demonstrated that L. longipalpis saliva is able to both increase the percentage of macrophages that became infected with Leishmania in BALB/c and C3H/HeN mice and exacerbate the parasite load in these cells [41]. The authors discuss the possibility that, during natural transmission, saliva could reduce the promastigote exposure to the immune system by attracting host cells to the bite site and by accelerating the uptake of these parasites. Exploring a straightforward and consistent model—the mouse air pouch—to investigate the inflammatory response induced by L. longipalpis, our group has described that L. longipalpis salivary gland sonicate was able to induce not only macrophages, but also neutrophil and eosinophil recruitment after 12 h in BALB/c [42]. The increased macrophage recruitment was linked to production of chemokine CCL2/MCP-1 and expression of its receptor CCR2 in the air pouch lining tissue. It was observed that L. longipalpis also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation [42]. This is noteworthy because it increases the availability of “safe targets,” the macrophages, for parasite evasion of the effector immune responses [43]. Interestingly, the recruitment profile observed in BALB/c was not observed in C57BL/6 mice, indicating that the same salivary components can induce diverse inflammatory effects depending on the host background [42]. However, because of limited number of cells that can be recovered on the air pouch model, some questions concerning early inflammatory events could not be investigated. Alternatively, the peritoneal cavity has been employed to this kind of study allowing the collection of high number of immigrating cells [44, 45]. In this regard, leukocyte recruitment into peritoneal cavity induced by L. longipalpis saliva has been evaluated in both BALB/c and C57BL/6 mouse strains [45]. In this work, significant neutrophil recruitment was observed six hours after administration of saliva, L. major, or saliva plus L. major. However, in BALB/c mice, all stimuli were able to induce more neutrophil migration than in C57BL/6 mice. Seven days later, it was observed that all stimuli were able to induce higher numbers of eosinophils and mononuclear cells in BALB/c when compared with C57BL/6 mice [45]. This study focused on the effect of saliva from L. longipalpis on adaptive immunity, evaluating CD4+ T lymphocyte migration and production of IL-10 and IFN-γ cytokines [45]. 4.1. Inflammatory Events Triggered by L. longipalpis Saliva. Neutrophils rapidly accumulate at the inflammatory site (as reviewed in [46]) and have been described on the sand fly bite site [23, 40]. Focusing on inflammatory events triggered by L. longipalpis saliva using the peritoneal model, we could observe a distinct kinetic of neutrophil recruitment to the peritoneal cavity of BALB/c and C57BL/6 mice (Figure 2). A late neutrophil influx was observed in BALB/c mice (Figure 2(a)), whereas in C57BL/6 mice neutrophils were 5 already evident in the first hours after L. longipalpis saliva inoculation compared to mice injected with endotoxin-free saline (Figure 2(b)). The link between neutrophil recruitment induced by L. longipalpis saliva and other events which initiate and switch off the inflammatory response is an attractive field to be explored. Inflammation resolution is regulated by the release of mediators that contribute to an orchestrated sequence of events [47]. For simplicity, they result in predominance of neutrophils in the inflamed area which are later replaced by monocytes that differentiate into macrophages. During the resolution, inflammatory cells undergo apoptosis and are phagocytosed. Clearance of apoptotic cells by macrophages drives a response characterized by release of antiinflammatory mediators [48]. Such safe removal of apoptotic cells has been implicated in exacerbation of Leishmania infection [49, 50]. The influence of L. longipalpis saliva in the time course of inflammation could be observed in cytospin preparations of the peritoneal cells from C57BL/6 mice. Neutrophils in contact with or phagocytosed by macrophages were observed at six hours (Figures 2(c) and 2(d)) and leukocyte phagocytosis by macrophages was an early event as well (Figure 2(e)). Moreover, apoptotic neutrophils were evident in C57BL/6 mice in the presence of saliva (Figure 2(f)). Therefore, components of sand fly saliva are able to both recruit and induce proapoptotic effects on neutrophils. These findings, in the scenario of anti-inflammatory clearance of apoptotic cells, add to the notion of beneficial effects of vector saliva on Leishmania transmission. Further work on mediators and mechanisms involved in this process is necessary. 5. Host Macrophage Response to L. longipalpis Saliva Sand fly saliva displays an important role in the macrophage response by triggering the recruitment [42, 51] and suppressing the killing of parasites within macrophages [41, 52]. In this regard, P. papatasi saliva inhibits the NO production in macrophages treated with IFN-γ [52] and L. longipalpis saliva hampers Leishmania antigen presentation to T lymphocytes by macrophages [53] as well as upregulates the IL-10 production related with NO suppression in macrophages infected with L. amazonensis [54]. Moreover, pure adenosine from P. papatasi saliva decreases NO production in murine macrophages [55] and maxadilan peptide present in L. longipalpis saliva upregulates IL-6, IL-10, and TGF-β cytokine responses of LPS-activated macrophages and downregulates IL-12, TNF-α, and NO associated with L. major killing [56]. Despite this, few research reports cover the cellular pathways involved in sand fly saliva modulation of macrophage response. Previous study showed that maxadilan acts on PAC-1 receptor in LPS-activated macrophages and inhibits TNF-α production whereas it increases IL-6 and PGE2 [11], and the authors suggest the participation of cAMP activation by maxadilan in this process. Although the literature abounds with reports on the effects of sand fly saliva in the immune response and infection, 49 6 Journal of Parasitology Research (a) (e) 4 Leukocytes/mφ (%) 5 PMN (×104 /mL) (c) BALB/c 4 3 ∗∗ 2 1 ∗ 2 1 0 0 6 12 24 (hours) ∗∗ 3 48 3 6 12 24 (hours) Saline SGS (b) ∗∗ 4 6 ∗∗ 3 2 1 0 6 (f) Pyknotic nuclei (%) PMN (×104 /mL) 5 (d) C57BL/6 12 24 48 (hours) ∗ 5 4 3 2 1 0 3 6 (hours) 24 Saline SGS Figure 2: Neutrophil influx, apoptosis, and phagocytosis into BALB/c and C57BL/6 peritoneal cavity in response to L. longipalpis saliva. Mice were injected with endotoxin-free saline or L. longipalpis salivary gland sonicate (SGS) (0.5 pair/animal). After stimulation, peritoneal cavities were washed and differential cell counts were performed on Diff-Quik stained cytospin preparations. (a-b) Kinetics of neutrophil recruitment in BALB/c (a) and C57BL/6 (b) mice. (c-d) Representative events of C57BL/6 neutrophil phagocytosis by macrophages on DiffQuik stained cytospin (magnification 1000x). (e-f) Phagocytosis of C57BL/6 leukocytes by macrophages (e) and neutrophil apoptosis (f) after stimulation with SGS (•) or saline (). Data shown are from a single experiment representative of three independent experiments. Values represent means ± SEM of five mice per group. ∗ P < 0.05 and ∗∗ P < 0.01. the effect of whole sand fly saliva on macrophages is poorly understood. Recently, we showed that L. longipalpis saliva activates lipid body (LB) formation in resident macrophages committed with PGE2 production by COX-2 enzyme (Figure 3) [51]. Lipid bodies are intracellular sites related with eicosanoid production, and their formation can be triggered by activation via different intracellular pathways (as reviewed in [57]). In this context, L. longipalpis saliva activated ERK-1/2 and PKC phosphorylation and the inhibition of both pathways resulted in blockade of saliva-induced PGE2 production by macrophages [51]. PGE2 modulates the macrophage response during Leishmania infection in macrophages [58, 59] and is related with parasite dissemination after infection; however, the role of saliva in the PGE2 released by macrophages during Leishmania infection remains to be addressed. Further studies will be necessary to clarify the importance of eicosanoids stimulated by sand fly saliva in macrophage clearance of parasites and consequently in parasite transmission after sand fly bite. 6. Neutrophils and L. longipalpis Saliva: A Neglected Interaction on Scenery of Leishmania Infection Looking to the neutrophils as a significant host-defense cell player in both innate and adaptive response of immune system, it is surprising that few works have attempted to investigate the consequences of vector’s saliva and neutrophils interaction in the pathogenesis of leishmaniasis. The reasons to encourage this special attention rise from several lines of evidence showing that neutrophils participate in Leishmania immunopathogenesis, by uptaking promastigote forms, producing cytokines and inflammatory mediators or interacting with macrophages enhancing infection (as reviewed in [60, 61]). Neutrophils are considered as an initial target of Leishmania infection [40, 62], and they are implicated in the immunopathogenesis of murine leishmaniasis [50, 63, 64]. Moreover, significant numbers of neutrophils are present at 50 Journal of Parasitology Research 7 Saliva Mφ recruitment Saliva ↑MCP-1 P-ERK-1/2 ↑PGE2 P-PKC-a ↑FasL ↓ROS COX-2 LBs PGE2 Macrophage Apoptosis via caspase Increase parasite burden Neutrophil Figure 3: Effects of Lutzomyia longipalpis saliva on macrophage activation and neutrophil apoptosis. Macrophages and neutrophils are the first host cells to contact Leishmania after sand fly bite. Saliva triggers macrophages activation by lipid bodies formation committed with the PGE2 production via COX-2 after phosphorilation of kinases. On the other hand, saliva induces neutrophil apoptosis by caspase and FasL activation. In addition, neutrophils activated by saliva become susceptible to Leishmania chagasi and release MCP-1, which is associated with macrophage recruitment. This scenario promoted by L. longipalpis saliva can contribute to Leishmania transmission in the early times of infection. the inoculation site, lesions, and draining lymph nodes from Leishmania-infected mice [31, 63, 65–67]. In addition, Leishmania parasites undergo a silent entry into macrophages inside phagocytosed neutrophils, thus reinforcing the role of neutrophils on establishment of Leishmania infection [68]. Leishmania donovani inhibition of traffic into lysosomederived compartments in short-lived neutrophils was suggested as a key process for the subsequent establishment of long-term parasitism [69]. On the other hand, neutrophils have also been implicated in parasite control. Phagocytosis of L. major by human neutrophils led to parasite killing [70]. Human neutrophils were capable to kill L. donovani by oxidative mechanisms [71], and, more recently, it was described the involvement of NET’s (Neutrophil Extracellular Traps) on L. amazonensis destruction [72]. One elegant approach that reinforced the essential role for neutrophils in leishmaniasis revealed the presence of Leishmania-infected neutrophil on the sand fly bite site [40]. However, in that work, although the sustained neutrophil recruitment had been evident only in response to the sand fly bite, the authors did not attribute the neutrophil influx to vector salivary components. Surprisingly, besides neutrophil recruitment, there are no previous reports on further effects of sand fly saliva on neutrophil inflammatory response. Interestingly, studies performed with tick saliva disclose that the inhibition of neutrophil functions favors the initial survival of spirochetes [73–75]. Our group has recently shown the first evidence of direct effect of L. longipalpis salivary components on C57BL/6 mice neutrophils [76]. In summary, we described that saliva from L. longipalpis triggers apoptosis of inflammatory neutrophils obtained from C57BL/6 peritoneal cavity (Figure 3). The proapoptotic effect of saliva was due to caspase activation and FasL expression on neutrophil surface. Although salivary glands from blood feeding vectors have a variety of components [76], it seems that the proapoptosis compound in L. longipalpis saliva is a protein. However, further work is required to elucidate which protein or proteins act in this process. Additional helpful information from this study is that preincubation of L. longipalpis saliva with anti-saliva antibodies abrogated neutrophil apoptosis. This allows us to propose that proapoptotic component from L. longipalpis saliva could be target for the host’s antibodies. Moreover, neutrophil apoptosis induced by L. longipalpis saliva was also increased in the presence of L. chagasi [76]. This is particularly interesting by reinforcing the synergistic effect of both vector component and parasite on host inflammatory response, as have been observed in cell chemotaxis [42]. Interestingly, saliva from L. longipalpis enhanced L. chagasi viability inside neutrophils. This effect was attributed to modulation of neutrophil inflammatory response [76], as treatment of neutrophils with a pan caspase inhibitor (z-VAD) and a COX-2 inhibitor (NS398) abrogated the increased parasite burden observed. Finally, we also described a novel inflammatory function of L. longipalpis saliva on neutrophils, stimulating MCP1 production, able to attract macrophages in vitro. Even though chemotatic activity from L. longipalpis saliva has been previously reported, this is the first demonstration that saliva modifies directly the neutrophil inflammatory function, inducing the release of chemotatic factors by these cells. 51 8 7. Future Directions In this paper, we explored the new inflammatory events induced by L. longipalpis in the recruitment and cellular function of leukocytes, as well as the repercussion to L. chagasi infection. The understanding of protective mechanisms regarding the initial steps of host’s response to salivary molecules that can correlate with resistance or susceptibility to Leishmania has been poorly explored. Further investigation should address factors that determine the success of Leishmania infection. Identifying new escape mechanisms used by Leishmania associated to the pharmacological complexity of the sand fly saliva remains a challenge. In this scenario, phylogenetic implications between vector and Leishmania species can result in distinct action under host cells. The insights from the inflammatory scenery approached here, as lipid body induction in macrophages and apoptotic death of neutrophils, need to be investigated during the interaction between saliva from other sand fly and Leishmania species. Another important point is that these inflammatory effects were detected in salivary gland extract of sand fly vector. However, recombinants proteins from L. longipalpis saliva that presented known immunogenic role should be tested as inducers of these inflammatory events during infection by Leishmania sp. The studies discussed here suggest that saliva components can act on virulence factors from parasite surface in the first steps involved the recognition, resistance to oxidative mechanisms, and modulation of inflammatory mediators’ produced by host cells. However, this finding seems to be part of a “large puzzle,” since they are viewed in isolation, by methodological limitations. Recent emerging imaging technologies have opened the possibility to monitor the process of Leishmaniahost cell interaction in real time from the first moment upon sand fly bite, allowing understanding of molecular and cellular mechanisms in Leishmania experimental infection. 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Luz et al., “Lutzomyia longipalpis saliva drives apoptosis and enhances parasite burden in neutrophils,” Journal of Leukocyte Biology, vol. 90, no. 3, pp. 575–582, 2011. 11 55 4.3. MANUSCRITO III Lutzomyia longipalpis Saliva Favors Leishmania infantum chagasi Infection Through Modulation of Eicosanoids A Saliva de Lutzomyia longipalpis Favorece a Infecção por Leishmania infantum chagasi Através da Modulação de Eicosanoides Este trabalho avalia o efeito da saliva no modelo peritoneal murino de macrófagos quanto à formação de corpúsculos lipídicos e a produção de eicosanoides associada a essas organelas, bem como vias de sinalização envolvidas neste processo. Resumo dos resultados: Neste trabalho, nós avaliamos o efeito da saliva de Lutzomyia longipalpis sobre a produção de eicosanoides durante os momentos iniciais da infecção por L. i. chagasi no modelo peritoneal murino. Nós observamos que a saliva aumentou a viabilidade intracelular de L. i. chagasi tanto em monócitos como em neutrófilos recrutados para a cavidade peritoneal. As células recrutadas para cavidade peritoneal apresentaram maiores níveis da relação PGE2/LTB4 e o pré-tratamento com NS-398, o inibidor de COX-2, reverteu o efeito da saliva sobre a viabilidade intracelular dos parasitas. Este artigo será submetido ao periódico internacional Parasites & Vectors (Fator de impacto JCR 2011 = 2.937). 56 1 Lutzomyia longipalpis Saliva Favors Leishmania infantum chagasi Infection Through 2 Modulation of Eicosanoids 3 4 Théo Araújo-Santos1,2, Deboraci Brito Prates1,3, Jaqueline França-Costa1,2, Nívea Luz1,2, 5 Bruno B. Andrade4, José Carlos Miranda1, Claudia I. Brodskyn1,2,5, Patrícia T. Bozza6, Aldina 6 Barral1,2,5 and Valéria Matos Borges1,5* 7 8 1. Gonçalo Moniz Research Center, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, BA, 9 Brazil; 10 2. Federal University of Bahia (UFBA), Salvador, BA, Brazil; 11 3. Departamento de Biomorfologia, Instituto de Ciências da Saúde, Universidade Federal da 12 Bahia, 40110-100 Salvador, BA, Brazil; 13 4. Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and 14 Infectious Diseases, National Institutes of Health, 20893, Bethesda, MD, USA; 15 5. Institute for Investigation in Immunology, iii-INCT (National Institute of Science and 16 Technology), São Paulo, Brazil. 17 6. Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil; 18 19 * Correspondence: [email protected] 20 Phone: +55 71 3176-2215. Fax: +55 71 3176-2279. 21 22 Running Title 23 Leishmania escape by saliva-driven eicosanoid 24 25 57 26 Abstract 27 Monocytes and neutrophils are considered the first line of host defense against infections, and 28 seem to be implicated in the immunopathogenesis of leishmaniasis. Here, we evaluated the 29 effect of Lutzomyia longipalpis salivary gland sonicate (SGS) on neutrophil and monocyte 30 recruitment and activation of eicosanoid production in a murine model of inflammation. 31 Intraperitoneal injection of L. longipalpis SGS together with L. i. chagasi induced an early 32 increased parasite viability inside monocytes and neutrophils. L. longipalpis SGS increased 33 PGE2 but reduced LTB4 production ex vivo in peritoneal leukocytes. In addition, the 34 pharmacological inhibition of COX-2 with NS-398 decreased parasite viability during 35 Leishmania infection in the presence of L. Longipalpis SGS indicating that PGE2 is important 36 to prevent parasite killing. These findings point out L. longipalpis SGS as a critical factor 37 driving immune evasion of Leishmania through modulation of eicosanoids, which may 38 represent an important mechanism on establishment of the infection. 39 40 Introduction 41 42 Despite of efforts towards the development of an antileishmanial vaccine and effective 43 antiparasite agents, visceral leishmaniasis (VL) continues to cause high morbidity and 44 considerable mortality worldwide (WHO, 2002). In America VL is transmitted by the bite of 45 Lutzomyia longipalpis sand flies. Transmission of Leishmania sp. by hematophagous sand fly 46 vectors occurs during blood feeding, when salivary content is inoculated alongside 47 Leishmania into host skin. Sand fly saliva enhances Leishmania infection on several 48 experimental models [1–3] through its modulatory effects on the host immune system [4,5]. A 49 successful blood feeding depends on the formation of a blood hemorrhagic pool [6]. In such 50 microenvironment there are many inflammatory cells [4], and L. longipalpis saliva has been 58 51 shown to enhance recruitment of immune cells, including monocytes and neutrophils [7–9]. 52 Macrophage recruitment induced by L. longipalpis saliva has been previously described by 53 our group using the air pouch model. However, restrictions related to this model make it 54 impossible to point out a more detailed effect of saliva in the leukocytes recruited. The 55 peritoneal cavity is a self-contained and delineated compartment [9], and for this reason, 56 several studies have described the use of the peritoneal model to investigate the leukocyte 57 migration induced by sand fly salivary gland extracts [9–11] as well as by Leishmania 58 [9,12,13]. We have previously shown that L. longipalpis salivary gland sonicate (SGS) is able 59 to modulate eicosanoid release in monocytes and neutrophils recruited to peritoneal cavity 60 [14]. In neutrophils, SGS benefits L. i. chagasi infection stimulating production of PGE2 in 61 vitro [15]. 62 In the present study, we explore the effect of L. longipalpis SGS on the eicosanoids 63 production in the context of L. i. chagasi infection in vivo using the peritoneal model in mice. 64 In addition, we demonstrate that eicosanoids can be important in modulation of immune 65 response elicited by SGS allowing increase in parasite viability as well as burden during early 66 moments of L. i. chagasi infection. 67 68 Methods 69 70 Antibodies and Reagents 71 Schneider’s insect medium, N-(1-naphthyl)-ethylenediamine and p-Aminobenzene- 72 sulfanilamide were purchased from SIGMA (St. Louis, MO). RPMI 1640 medium and L- 73 glutamine, penicillin, and streptomycin were from Invitrogen (Carlsbad, CA, USA). 74 Nutridoma-SP was from Roche (Indianapolis, In, USA). A23187 calcium ionophore was from 75 Calbiochem Novabiochem Corp. (La Jolla, CA). NS-398, PGE2 and LTB4 enzyme-linked 59 76 immunoassay (EIA) Kits were from Cayman Chemical (Ann Arbor, MI). Dimethylsulfoxide 77 (DMSO) was purchased from ACROS Organics (New Jersey, NJ). 78 79 Animals 80 Inbred male C57BL/6 mice, age 6–8 weeks, were obtained from the animal facility of Centro 81 de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz (CPqGM-FIOCRUZ, Bahia, Brazil). 82 The animals were kept at a temperature of 24 °C, with free access to food and water and light 83 and dark cycles of 12 hours each. 84 85 Ethics Statement 86 All experiments were performed in strict accordance with the recommendations of the 87 Brazilian National Council for the Control of Animal Experimentation (CONCEA). The 88 Ethics Committee on the use of experimental animals (CEUA) of the Centro de Pesquisas 89 Gonçalo Moniz, Fundação Oswaldo Cruz – (Permit Number: 27/2008) approved all protocols. 90 91 Parasite 92 L. i. chagasi (MCAN/BR/89/BA262) promastigotes were cultured at 25°C in Schneider’s 93 insect medium supplemented with 20% inactive FBS, 2 mM L-glutamine, 100 U/ml 94 penicillin, and 100 µg/ml streptomycin. 95 96 Sand flies and preparation of salivary glands 97 Adult Lutzomyia longipalpis captured in Cavunge (Bahia, Brazil) were reared at the 98 Laboratório de Imunoparasitologia/CPqGM/FIOCRUZ (Bahia, Brazil) as described 99 previously [8]. Salivary glands were dissected from 5- to 7-day-old L. longipalpis females 100 under a stereoscopic microscope (Stemi 2000, Carls Zeiss, Jena, Germany) and stored in 60 101 groups of 10 pairs in 10 µl endotoxin-free PBS at -70°C. Immediately before use, glands were 102 sonicated (Sonifier 450, Brason, Danbury, CT) and centrifuged at 10,000 x g for 4 minutes. 103 The supernatants of salivary gland sonicate (SGS) were used for the experiments. The level of 104 LPS contamination of SGS preparations was determined using a commercially available LAL 105 Chromogenic Kit (QCL-1000, Lonza Bioscience) resulting in negligible levels of endotoxin 106 in the salivary gland supernatant. All experimental procedures used SGS equivalent to 0.5 107 pair of salivary gland per group which possesses approximately 0.7 micrograms of proteins 108 [16]. 109 110 Mice infection 111 C57BL/6 mice were submitted to intra-peritoneal (i.p.) injection of with 0.1 ml of SGS (0.5 112 pair/cavity), 0.1 ml of L. i. chagasi (3x106/cavity), 0.1 ml of endotoxin-free saline per cavity 113 (negative control) or 0.1 ml of LPS (20µg/ml; positive control). One hour post stimulus the 114 total leukocytes that migrated to the peritoneal cavity was harvested by peritoneal lavage with 115 injection of 10 ml endotoxin-free saline. Alternatively, C57BL/6 mice were previously treated 116 with an i.p. injection of NS398 2 mg/kg or DMSO as a vehicle control. Total counts were 117 performed on a Neubauer hemocytometer after staining with Turk’s solution. Differential cell 118 counts (200 cells total) of infected cells were carried out microscopically on cytospin 119 preparations stained with Diff-Quick. 120 121 Assessment of intracellular load of L. i. chagasi 122 Intracellular load of L. i. chagasi was estimated by production of proliferating extracellular 123 motile promastigotes in Schneider medium [17]. Briefly, after 1h of infection, peritoneal cells 124 were centrifuged, supernatants containing non-internalized promastigotes were removed and 125 medium was replaced by 250 µl of Schneider medium supplemented with 20% inactive FBS, 61 126 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Infected cells were 127 cultured at 25°C for additional 3 days. In the third day promastigotes in the cultures were 128 counted in a Neubauer hemocytometer. 129 130 Transmission Electron Microscopy 131 Peritoneal cells from mice infected with L. i. chagasi were centrifuged (1500 rpm, 10 min) 132 and the pellets were resuspended and fixed in a mixture of freshly prepared aldehydes (1% 133 paraformaldehyde and 1 % glutaraldehyde) in 0.1 M phosphate buffer, pH7.4 overnight at 134 4°C. The cells were washed in the same buffer and embedded in molten 2% agar (Merk). 135 Agar pellets containing the cells were post-fixed in a mixture of 1% phosphate-buffered 136 osmium tetroxide and 1.5% potassium ferrocyanide (final concentration) for 1 h and 137 processed for resin embedding (PolyBed 812, Polysciences, Warrington, PA). The section 138 were mounted on uncoated 200-mesh copper grids and viewed with a transmission electron 139 microscope (EM 109; Zeiss, Germany). Electron micrographs were randomly taken at the 140 magnifications of 7 000 – 30 000X to study the entire cell profile. 141 142 PGE2 and LTB4 measurements 143 PGE2 and LTB4 levels were measured ex vivo from leukocytes harvested by peritoneal cavity 144 washing with Ca2+-Mg2+-free HBSS. After, recovered cells (1x106 cells/ml) were 145 ressuspended in HBSS contained Ca2+-Mg2+ and then stimulated with A23187 (0.5 µM) for 146 15 min. Reactions were stopped on ice, and samples were centrifuged at 500 x g for 10 min at 147 4°C. Supernatants were collected to measure PGE2 and LTB4 by enzyme-linked immunoassay 148 (EIA), according to manufacturer’s instructions. 149 150 Statistical analysis 62 151 Each experiment was repeated at least three times. The data are presented as the mean and 152 SEM (standard error) of representative experiments and were analyzed using the GraphPad 153 Prism 5.0 software (GraphPad Software, San Diego, CA, USA). The comparisons between 154 two groups were analyzed using Mann-Whitney test. The differences were considered 155 statistically significant when p ≤ 0.05. 156 157 Results 158 L. longipalpis SGS enhances parasite viability during L. i. chagasi infection in vivo 159 We have previously shown that the main cell types recruited to the inoculation site by L. 160 longipalpis saliva are neutrophils [8] and macrophages [7,14]. Here we observed this event 161 early (Supporting Information fig. S1) and curiously, when inoculated together L. i. chagasi, 162 SGS does not alter the number of infected neutrophils (fig. 1A) or monocytes (fig. 1B) 163 recovered from the peritoneum of mice injected with L. i. chagasi plus SGS after 1 hour. 164 However, significant increase in the number of viable parasites were obtained from cultures 165 of peritoneum recovered cells after the same time (fig. 1C). We have also evaluated the 166 presence of parasites inside peritoneal neutrophils and monocytes by electron microscopy 167 (fig. 2A-D). Cells recovered from mice injected with L. i. chagasi alone frequently presented 168 degenerated parasite inside vacuoles from neutrophils (fig. 2A) and monocyte (fig. 2B). In 169 contrast, when L. longipalpis SGS was inoculated together with Leishmania, viable parasites 170 were recurrently observed in both neutrophils (fig. 2C) and monocytes (fig. 2D), although the 171 relative number of these leukocytes were not enhanced by SGS plus L. i. chagasi inoculum 172 (fig. S1). 173 174 Effect of L. longipalpis SGS on eicosanoids production and L. i. chagasi infection ex vivo 175 It has been well demonstrated that different classes of eicosanoids promote both cellular 176 recruitment and safe removal of inflammatory cells coordinating the initial events of 63 177 inflammation [18]. In addition, LTB4 is important in host responses to infection because it 178 enhances leukocyte accumulation and phagocytic capacity [19]. We have previously 179 demonstrated that L. longipalpis is able to recruit neutrophils [20] and monocytes [14] to 180 peritoneal cavity and increases PGE2 but not LTB4 in these cells [14]. Considering these 181 findings, our further interest was to investigate whether L. longipalpis saliva is involved in 182 augmenting of PGE2 and LTB4 production by L. i. chagasi-infected mice. We measured PGE2 183 and LTB4 levels on supernatants of peritoneal cells recovered 1 hour after injection with L. i. 184 chagasi in the presence or absence of L. longipalpis SGS. Addition of SGS to Leishmania did 185 not alter PGE2 levels by peritoneal cells (fig. 3A) while LTB4 production was dramatically 186 reduced (fig. 3B). Concerning the fact that PGE2 and LTB4 present antagonistic effects on 187 inflammation and Leishmania infection [21,22], we addressed the inflammatory status of this 188 dynamic process by plotting the PGE2/LTB4 ratio (fig. 3C). L. longipalpis salivary 189 components triggered high PGE2/LTB4 ratio 1 h after stimulation (fig. 3C). Based on these 190 observations we hypothesized that L. longipalpis SGS favors L. i. chagasi infection by 191 inhibiting LTB4 production and favoring PGE2 formation. To assess if the manipulation of the 192 eicosanoid balance driven by SGS is important to L. i. chagasi infection in vivo, we inhibited 193 pharmacologically the PGE2 production by using a cicloxigenase-2 (COX-2) selective 194 inhibitor NS398. The inhibition of COX-2 decreased number of viable parasites in peritoneal 195 cells after L. i. chagasi infection in the presence of SGS (fig. 4). 196 197 Discussion 198 Sand fly saliva displays an important role in the first steps of Leishmania infection. In this 199 regard, saliva induces cellular recruitment to inflammatory site, inhibits proinflammatory 200 cytokines and deactivates dendritic cells to mobilize regulatory T cells [5]. Previous studies 201 have shown the participation of eicosanoid in the inflammatory response triggered by sand fly 202 saliva [9,14,23]. Herein, we showed for the first time that saliva can modulate eicosanoid 64 203 profile with a balance skewed towards COX-2 driven PGE2 over LTB4 during early time post 204 L. i. chagasi inoculation, benefiting infection. 205 PGE2 production supports establishment of several pathogen infections [24]. In rats and mice, 206 Trypanossoma cruzi infection induces PGE2 by macrophages [25–27]. During Mycobacterium 207 bovis infection, the increase of PGE2 and TGF-β1 production by macrophages that phagocyte 208 apoptotic neutrophils in the inflammatory site increases infection [28]. In addition, the 209 interaction between human apoptotic neutrophils and macrophages also increases L. 210 amazonensis infection via PGE2 and TGF-β1 production [29]. On the other branch of the 211 inflammatory response is LTB4. The production of LTB4 is associated to increase of pathogen 212 killing [19,30]. In the context of Leishmania infection, LTB4 is involved in nitric oxide 213 production and reduced parasite burden in susceptible and resistant mice to L. amazonensis 214 [21]. We have previously shown that L. longipalpis saliva promptly activates macrophages to 215 produce PGE2 but not LTB4 [14] in vitro and ex vivo. In addition, SGS increases PGE2 216 production by neutrophils during L. i. chagasi infection [15]. Here we demonstrate that L. 217 longipalpis SGS reduce the early LTB4 production during L. i. chagasi, infection whereas 218 orchestrates an anti-inflammatory response by increment of PGE2 production. In addition, the 219 inoculation of L. longipalpis SGS plus L. i. chagasi increased parasite viability inside 220 peritoneal cells. The pharmacological inhibition of COX-2 reversed the effect of SGS on 221 enhancing parasite viability. 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J Leukoc Biol 84: 389– 324 396. 69 325 30. Serezani CH, Aronoff DM, Jancar S, Mancuso P, Peters-Golden M (2005) 326 Leukotrienes enhance the bactericidal activity of alveolar macrophages against 327 Klebsiella pneumoniae through the activation of NADPH oxidase. Blood 106: 1067– 328 1075. 329 330 331 Figure legends 332 333 Figure 1. L. longiplapis SGS favors L. i. chagasi survival inside neutrophils and 334 monocytes. C57BL/6 mice were inoculated with L. i. chagasi and/or SGS according to 335 methods. Percentage of infected (A) neutrophils and (B) monocytes were estimated on Diff- 336 Quick-stained cytospin preparations. (C) The figure shows viable parasite counting recovered 337 by total infected peritoneal cells. Bars represent the mean ± SEM, n = 3. p values are shown 338 on graphs. 339 340 Figure 2. L. longiplapis SGS favors viability of L. i. chagasi inside neutrophils and 341 monocytes. C57BL/6 mice were inoculated with L. i. chagasi and/or SGS according to 342 methods. Transmission electron microscopic images of peritonial cells after 1h infection with 343 L. i. chagasi are shown. Disrupted L. i. chagasi inside neutrophils (A) and monocytes (B) are 344 showed. Viable parasites were observed in neutrophils (C) and monocytes (D) those animals 345 infected in the presence of L. longipalpis SGS. Insets indicated by white arrowheads shows 346 details of parasite inside parasitophorous vacuoles (PV) outlined in white (50k-fold 347 increase). P-parasite. 348 70 349 Figure 3. Eicosanoid production in response to L. longipalpis SGS during L. i. chagasi 350 infection. C57BL/6 mice were injected i.p. with saline (control), L. i. chagasi and/or SGS 351 according to methods. One hour after stimulation, peritoneal cavities were washed and cells 352 were harvested. The cells were then incubated with A23187 (0.5 mM) for 15 min at 37ºC to 353 evaluate LTB4 and PGE2 production. The concentrations of PGE2 (A) and LTB4 (B) in the 354 supernatant were measured by ELISA. (C) The figure shows the PGE2/LTB4 ratios. The data 355 are the means and SEM from an experiment representative of three independent experiments. 356 p values are showed on graphs. 357 358 Figure 4. Eicosanoid inhibition affects the parasite viability in vivo during L. i. chagasi 359 infection in the presence of SGS. C57BL/6 mice were treated with DMSO (vehicle – Veh), 360 NS398 2 mg/kg. After 1h of treatment, mice were injected i.p. with L. i. chagasi and SGS 361 according to methods. Graph shows viable parasite counting recovered by total infected 362 peritoneal cells. The data are the means and SEM from an experiment representative of three 363 independent experiments. 364 365 Supporting Information 366 Figure S1. Leukocyte recruitment in response to L. longipalpis SGS during L. i. chagasi 367 infection. C57BL/6 mice were injected i.p. with saline (control), L. i. chagasi and/or SGS 368 according to methods. One hour after stimulation, peritoneal cavities were washed and cells 369 were harvested. (A) Total leucocytes, (B) monocytes and (C) neutrophil were estimated on 370 Diff-Quick-stained cytospin preparations. The data are the means and SEM from an 371 experiment representative of three independent experiments. p values are showed on graphs. 372 71 373 Figure 1 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 Figure 2 72 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 Figure 3 73 423 Figure 4 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 Figure S1 74 4.4. MANUSCRITO IV Prostaglandin F2α Production in Lipid Bodies from Leishmania infantum chagasi is a Critical Virulence Factor A Produção Prostaglandina F2α em Corpúsculos Lipídicos de Leishmania infantum chagasi é um crítico fator de virulência Durante os estudos anteriores, ao tentarmos avaliar a formação de CLs em células infectadas observamos a presença de CLs restrita ao parasito Leishmania. Neste trabalho, nós caracterizamos a dinâmica de formação dos CLs de L. i. chagasi. Além disso, verificamos o papel dessa organela na produção de prostaglandina F2α pelo parasita e a importância deste eicosanoide durante a infecção de macrófagos. Resumo dos resultados: Neste estudo nós descrevemos a dinâmica de formação e a distribuição celular dos CLs nas distintas formas evolutivas de L. i. chagasi utilizando técnicas de microscopia ótica convencional, confocal e microscopia eletrônica de transmissão. Aqui, nós verificamos que a quantidade de CLs é aumentada durante a metaciclogênese. Além disso, a expressão de PGF2α sintase (PGFS) foi maior nas formas metacíclicas quando comparada às outras formas e a enzima foi localizada nos CLs. A adição de ácido araquidônico AA à cultura de Leishmania aumentou a quantidade de CLs por parasita, bem como os níveis de PGF2α nos sobrenadantes de cultura. A infecção com as diferentes formas de L. i. chagasi não foi capaz de estimular a formação de CLs na célula hospedeira. Entretanto, os parasitas intracelulares apresentaram maiores quantidades de CLs. A infecção estimulou uma rápida expressão de COX-2, mas não foi detectado aumento na produção de PGF2α nos sobrenadantes de 75 células infectadas. Por fim, nós verificamos a presença do receptor de PGF2α (FP) nos vacúolos parasitóforos e o pré-tratamento das células com um antagonista do receptor FP inibiu os índices de infecção de forma dose-dependente. Este artigo foi submetido ao periódico internacional PLoS Neglected Tropical Diseases (Fator de impacto JCR 2011 = 4.716) e encontra-se em segunda fase de revisão por pares. 76 1 Prostaglandin F2α Production in Lipid Bodies from Leishmania infantum chagasi is 2 a Critical Virulence Factor 3 Théo Araújo-Santos1,2,3, Nilda E. Rodríguez3,7, Sara de Moura Pontes1, 2, Upasna Gaur 4 Dixt3, Daniel R. Abánades4, Patrícia T. Bozza5, Mary E. Wilson3 and Valéria Matos 5 Borges1,2,6* 6 7 1. Gonçalo Muniz Research Center, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, 8 BA, Brazil; 9 2. Federal University of Bahia (UFBA), Salvador, BA, Brazil; 10 3. University of Iowa and the Iowa City VA Medical Center, Iowa City, IA, USA 11 4.Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, 12 C.S.I.C, Madrid, Spain 13 5. Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil. 14 6. Institute for Investigation in Immunology, iii-INCT (National Institute of Science and 15 Technology), São Paulo, Brazil 16 7. Present address, Department of Biology, University of Northern Iowa, Cedar Falls, 17 IA, USA 18 * Correspondence: [email protected] 19 Phone: +55 71 3176-2215. Fax: +55 71 3176-2279. 20 77 21 Footnote Page 22 The authors declare they have no commercial association that might pose a conflict of 23 interest. 24 25 Running Title 26 Lipid bodies from L. i. chagasi produce PGF2α 27 28 Abstract 29 Lipid bodies (LB) are cytoplasmic organelles involved in eicosanoid production in 30 leukocytes. Eicosanoids such as prostaglandins (PG) have been implicated in the 31 immune response control. Parasites such as Leishmania are also capable of producing 32 PGs, but the role of parasite LBs in biosynthesis of PGs has not yet been investigated. 33 In this work, we studied the dynamics of LB formation and PG release from Leishmania 34 infantum chagasi. Using light and electron microscopy techniques, we described here 35 the cellular arrangement and abundance of LBs during development of the protozoan L. 36 i. chagasi. In this regard, a virulent metacyclic state of Leishmania displayed more LBs 37 as well as expressed high levels of PGF2α synthase (PGFS) compared to others 38 developmental stages. Moreover, PGFS was localized in the parasite LBs and the 39 addition of exogenous arachdonic acid to procyclic Leishmania cultures increased 40 parasite LBs formation and PGF2α release. During macrophage infection with L. i. 41 chagasi, LBs were restricted to parasites inside the parasitophorous vacuoles (PV). 42 Notwithstanding, Leishmania infection upregulated COX-2 expression but this was not 43 followed by PGF2α release by macrophages. We detected PGF2α receptor (FP) on the 44 Leishmania PV surface by immunogold electron and fluorescence microscopy. The 45 blockage of FP receptor with AL8810, a selective antagonist, dramatically hampered 78 46 Leishmania infection suggesting that PGF2α should be important to parasite infectivity. 47 Overall these results suggest that PGF2α production in LBs is a virulence factor to 48 metacyclic forms of L. i. chagasi. The data demonstrate novel functions for LBs and 49 PGF2α in the cellular biology of Leishmania, with possible implications for interactions 50 with the surrounding host microenvironment. 51 52 Author Summary 53 Leishmania parasites contain the enzymes to synthesize prostaglandin F2α (PGF2α). It is 54 unknown whether PGF2α associates with lipid body (LB) formation in parasites, and 55 whether LB from the parasite and/or the host macrophage contribute to parasite 56 infectivity. We report here that LBs increased in abundance during development of the 57 protozoan L. i. chagasi to a virulent metacyclic state, as did the expression of PGF2α 58 synthase (PGFS). The abundance of parasite LBs, and of PGFS and PGF2α were 59 modulated by exogenous arachdonic acid, a substrate of PGFS. Infected macrophages 60 rapidly upregulate COX-2 expression but this was not followed by PGF2α release, 61 suggesting that the macrophage metabolites were used by parasites inside the 62 parasitophorous vacuole. Moreover, inhibition of the host PGF2α receptor dramatically 63 hampered Leishmania infection, suggesting that this prostaglandin may facilitate 64 parasite infectivity. The data demonstrate novel functions for prostaglandin F2α 65 production in LBs and for the PGF2α receptor (FP) in the cellular biology of Leishmania 66 with critical implications for the host-parasite interactions. 67 68 Introduction 69 Lipid bodies (also called lipid droplets) (LBs) are cytoplasmic organelles involved in 70 the storage and processing of lipids and are present in all cell types [1]. In leukocytes 79 71 and endothelial cells, LBs are critically involved in eicosanoid production because they 72 contain the necessary enzymatic machinery and substrates [2]. Several intracellular 73 pathogens take advantage of the LB formation in the host cells. The increase in the 74 number of host cell LBs and their recruitment to parasitophorous vacuoles have been 75 demonstrated in infections with Trypanossoma cruzi [3], Toxoplasma gondii [4], 76 Plasmodium falciparum [5], Chlamydia trachomatis [6] and Mycobacterium leprae [7] 77 The location of LBs close to phagolysosomes suggests that LBs could be used as a 78 source of nutrients by pathogens. In addition, an increase in the LB number in the 79 cytoplasm of macrophages is associated with release of PGE2 and enhancement of 80 M.bovis [8,9] and T. cruzi [3]. All together, these findings argue that induction of LB 81 formation by intracellular pathogens promotes their survival [10]. 82 Notwithstanding the morphological similarity between the LBs in leukocytes and 83 parasites, the function of parasite LBs and the eicosanoid production by its LBs have 84 not been demonstrated. Eicosanoids, such as prostaglandins (PG), are bioactive 85 molecules produced from arachidonic acid (AA) metabolism by specific enzymes, such 86 as cyclooxyganase (COX) and prostaglandin synthases. Prostaglandins have been 87 implicated in the control of immune responses [11,12]. Despite the absence of COX 88 genes and homologous proteins in the Order Trypasomatidae protozoa, parasites such 89 as Leishmania are capable of producing PGs [13]. These parasites contain the 90 prostaglandin F2α synthase (PGFS) responsible for PGF2α production [14]. PGF2α acts 91 directly on the PGF2α receptor (FP) and triggers the activation of the COX pathway 92 [15]. However, the question of whether PG biosynthesis localizes in parasite has not 93 been investigated. Beside is unknown what the role of PGF2α and your FP receptor in 94 the Leishmania-host interplay. 80 95 In this study, we investigated the dynamics of LB formation and PGF2α release in 96 Leishmania infantum chagasi (L. i. chagasi). In addition, we investigated the role of the 97 FP receptor in macrophages during L. i. chagasi infection. Our findings demonstrated 98 an increase in the expression of PGFS during L. i. chagasi metacyclogenesis and 99 showed that parasite-derived PGF2α plays a critical role in macrophage infection. 100 101 Materials and Methods 102 103 Antibodies and Reagents 104 The L-glutamine, penicillin, streptomycin, RPMI 1640 medium, Ca2+ Mg2+-free HBSS-/- 105 and HBSS+/+ with Ca2+ and Mg2+ were purchased from Gibco (Carlsbad, CA). 106 Dimethylsulfoxide (DMSO) was purchased from ACROS Organics (New Jersey, NJ). 107 The rabbit anti-FP receptor antibody, PGF2α enzyme-linked immunoassay (EIA) Kit and 108 AA were from Cayman Chemical (Ann Arbor, MI). The 4,4-difluoro-1,3,5,7,8- 109 pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) was obtained from 110 Molecular Probes (Eugene, OR) and osmium tetroxide (OsO4) was from Electron 111 Microscopy Science (Fort Washington, PA). Aqua-polymount was from Polysciences 112 (Warrington, PA). Thiocarbo-hydrazide and N-ethyl-N’- (3-dimethylaminopropyl) 113 carbodiimide hydrochloride (EDAC) were purchased from Sigma-Aldrich (St. Louis, 114 MO). Rat 1D4B anti-LAMP antibody was from the University of Iowa (Iowa City, IA). 115 The Texas Red-conjugated with goat anti-rabbit IgG and Vectashield H-1000 and 1200 116 medium were purchased from Vector Labs (Burlingame, CA). Alexa Fluor 647 and 117 488-conjugated with goat anti-rat IgG were purchased from Molecular Probes 118 (Carlsbad, CA). 81 119 120 Animals 121 Inbred male BALB/c mice, age 3–5 weeks, were obtained from the animal facility of 122 Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz (CPqGM-FIOCRUZ, 123 Bahia, Brazil). The animals were kept at a temperature of 24 °C, with free access to 124 food and water and light and dark cycles of 12 hours each. 125 126 Ethics Statement 127 All experiments were performed in strict accordance with the recommendations of the 128 Brazilian National Council for the Control of Animal Experimentation (CONCEA). The 129 Ethics Committee on the use of experimental animals (CEUA) of the Centro de 130 Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz – (Permit Number: 27/2008) 131 approved all protocols. 132 133 Wild-type Parasites 134 The Leishmania chagasi promastigotes (MHOM/BR/00/1669) were serially passed 135 through Syrian hamsters and isolated from spleens. Parasites were cultured in 136 hemoflagellate-modified minimal essential medium (HOMEM) containing 10% HI-FCS 137 for 7–9 days until the culture reached stationary phase. To obtain a pure population of 138 logarithmic-phase promastigotes, the cultures were re-diluted every 2 days for at least 3 139 consecutive cycles [16]. Metacyclic promastigotes were isolated from the stationary 140 cultures using the Ficoll-Hypaque (Sigma St. Louis, MO) density gradient separation 141 method described previously [17]. Amastigotes were isolated from the spleens of the 142 infected male Syrian hamsters and were incubated overnight h in amastigote growth 143 medium containing 20% FCS at 37ºC and 5% CO2, pH 5.5 [18]. 82 144 145 LcJ Parasites 146 The LcJ parasite line, derived from wild-type L. i. chagasi, converts between 147 promastigote and amastigote forms in axenic culture. LcJ promastigotes were 148 maintained in HOMEM, and amastigotes were maintained in a low pH medium with 149 fetal calf serum, as reported [19]. Parasites were switched from one stage to the other 150 every 3 weeks. To ensure that the LcJ promastigotes or amastigotes were fully 151 converted, the experiments were performed using parasites that were passaged three 152 times under conditions specific for each stage[18]. 153 154 Cloning, Expression and Purification of Prostaglandin F2α Synthase from L. i. 155 chagasi 156 The prostaglandin f2-alpha synthase/D-arabinose dehydrogenase (PGFS) coding region 157 (genedb code: LinJ31_V3.2210) was amplified from L. i. chagasi total DNA using the 158 polymerase chain reaction (PCR) and was cloned into the BamHI site of pBluescript 159 vector using the primers 5’-CGGGATCCATGGCTGACGTTGGTAAGGC-3’ and 5’- 160 CCAAGCTTTAGAACTGCGCCTCATCGGG-3’ (the restriction sites are underlined). 161 Amplification of the correct gene sequence was confirmed by DNA sequence analysis 162 (CPqGM – FIOCRUZ facility) and the coding region was subcloned in frame with an 163 N-terminal His6 tab in the pQE30 expression vector (Qiagen, Germany). 164 Expression of the recombinant protein was induced in E. coli cultures by the addition of 165 2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 h at 37ºC. The bacterial 166 lysates were loaded onto nitrilotriacetic acid (NTA) chromatographic columns, and the 83 167 protein purification was performed in accordance with the manufacturer’s instructions 168 (Qiagen, Germany). 169 Production of Antiserum Against L. i. chagasi Prostaglandin F2α Synthase 170 C57BL/6 mice were immunized intraperitoneally with 50μg of L. i. chagasi PGFS 171 recombinant protein in the presence of a 50% solution of Freud’s incomplete adjuvant 172 (Sigma-Aldrich) three times with a 15-day interval between each immunization. After 173 each immunization, mouse serum was collected and evaluated the anti-PGFS antibody 174 production using ELISA on plates coated with the PGFS recombinant protein (see 175 Figure S1A). The specificity of the antiserum to the L. i. chagasi PGFS protein was 176 evaluated by Western blot analysis of the L. i. chagasi total protein (see Figure S1B) as 177 described below. 178 179 Western Blotting 180 Leishmania parasites (2x108/mL) at different stages were lysed using LyseM solution 181 (Roche Mannheim, Germany). Sample protein concentrations were measured using the 182 BCA protein assay (Pierce, Rockford, IL). Total proteins (30μg) were separated by 10% 183 SDS–PAGE and were transferred to nitrocellulose membranes. The membranes were 184 blocked in Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TT) plus 5% 185 dry milk for 1 h before incubation overnight in murine anti-PGFS (1:1,000) antibodies. 186 After the removal of the primary antibody, the membranes were washed five times in 187 TT and were incubated in the peroxidase-conjugated secondary antibody (1:5,000) for 188 1h. The membranes were washed and developed using the ECL chemiluminscence kit 189 (Amersham, UK). The membranes were stripped in accordance with the manufacturer’s 190 instructions (Amersham, UK) and reprobed with primary anti-α tubulin (1:1,000) 84 191 antibody as a loading control. The protein bands were detected using the ImageQuant 192 LAS 4000 system (GE, Piscataway, NJ). 193 Culture and Infection of Bone Marrow Macrophages 194 Bone marrow cells were harvested from BALB/c mouse femurs and cultured at 37ºC 195 and 5% CO2 in RPMI-1640 medium supplemented with 10% HI-FCS, 2 mM L- 196 glutamine, 100 U/ml penicillin, and 50 mg/ml streptomycin (RP-10), and 20% L929 cell 197 culture supernatant (American Tissue Type Collection, Manassas, VA) as a source of 198 macrophage colony-stimulating factor. After 7–9 days, differentiated adherent bone 199 marrow derived macrophages (BMMs) were detached from the plate using 2.5 mg/ml 200 trypsin plus 1 mM EDTA (Gibco) [18]. Bone marrow macrophages (3x105/well) were 201 plated on coverslips in 24-well plates and cultured at 37ºC, 5% CO2 in RP-10 for 24 202 hours. 203 BMMs were either treated with 50, 10 and 1 µM of AL 8810 isopropyl ester or with 204 ethanol as the vehicle control. Treated macrophages were infected with non-opsonized 205 metacyclics promastigotes at a multiplicity of infection (MOI) of 10:1, LcJ 206 promastigotes at a MOI of 20:1 or LcJ amastigotes at a MOI of 3:1. Macrophage 207 binding was synchronized by centrifugation of BMMs and parasites for 3 min at 1,200 208 rpm and 4ºC, followed by placement at 37ºC, 5% CO2 at time = 0. 209 After 30 min extracellular parasites were removed by rinsing twice with HBSS without 210 Ca++ or Mg++ (HBSS-/-) followed by the addition of fresh RP-10. After specified times, 211 some coverslips were fixed, and stained with Diff Quik (Wright-Giemsa). Intracellular 212 parasites were counted under light microscopy. Other coverslips were harvested after 1, 213 4, 8, 24, 48 or 72 h, fixed in 2% paraformaldehyde and analyzed by confocal 214 microscopy as described below. 85 215 Measurement of PGFα production 216 Supernatants from Leishmania cultures medium or infected macrophages were collected 217 for measurement of PGF2α by enzyme-linked immunoassay (EIA) according to the 218 manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI). 219 COX-2 Expression 220 Total RNA was extracted from infected BMMs using RNeasy Protect Mini Kit (Qiagen, 221 USA) 1, 4 and 24 hours after infection. First-strand cDNA synthesis was performed 222 with 1µg of RNA in a total volume of 25 µL by using SuperScript II (Gibco, USA). 223 Oligonucleotide primers used were: GAPDH 5’-CTGACATGCCGCCCTGGAG-3’ and 224 3´-TCAGTGTAGCCCAGGATGCC-5’; COX-2 5’- 225 GCTCAGGTGTTGCACGTAGTCTT-3’ and 3’-TTCGGGAGCACAACAGAGTG-5’. 226 All primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa). 227 RT-PCRs were performed by using µ10 L Fast SYBR Green Master Mix in a total 228 volume of 20µL including cDNA samples and primers. The results were expressed by 229 ΔCt. 230 231 Confocal microscopy Analysis 232 Parasites were washed by centrifugation in HBSS-/- and subjected to cytospin onto glass 233 slides, fixed in 2% paraformaldehyde, permeabilized in 0.1% Triton X-100 for 10 min, 234 and rinsed with HBSS-/-. The parasites were incubated overnight in anti-PGFS 235 antiserum, and non-immune mouse serum as the negative control. 236 Infected macrophages were fixed in 2% paraformaldehyde and permeabilized with 0.1% 237 Triton X-100 in PBS for 15 min and blocked with 5% dry milk for 1 hour. To stain 86 238 parasitophorous vacuoles, BMMs were incubated with rat 1D4B anti-LAMP-1 (1:100) 239 in 5% milk/PBS overnight at 4°C, washed and incubated with secondary antibodies 240 (1:200) Alexa Fluor 647 or 488-conjugated with goat anti- rat IgG for 1h at room 241 temperature. 242 Both parasites and infected BMMs were stained for lipid bodies and nuclei. Cells were 243 first incubated in BODIPY® 493/503 (10 µM) at room temperature for 1h to stain the 244 lipid bodies. Cells were washed and then stained with 5ηg/mL ethidium bromide to 245 stain the nuclei. Images were analyzed by confocal microscopy using a Zeiss 510 246 microscope equipped with ZEN2009 software (Carl Zeiss, Inc., Thornwood, NY). 247 In addition, uninfected and infected macrophages were stained with anti-FP receptor 248 antibody (1:20) overnight at 4ºC, washed and incubated with Texas Red-conjugated 249 with goat anti-rabbit IgG for 1h at room temperature. The FP receptor staining was 250 colocalized with anti-LAMPI and DAPI staining (Vector Laboratories, Burlingame, 251 CA). Samples were observed by AX-70 Olympus microscopy and images were 252 acquired using the software Image-Pro Plus (Media Cybernetics, Silver Spring, MD). 253 254 Transmission Electron Microscopy 255 Metacyclic L. i. chagasi or infected BMMs were centrifuged, and the pellets were 256 resuspended and fixed in a mixture of freshly prepared aldehydes (1% 257 paraformaldehyde plus 1% glutaraldehyde) in 0.1 M phosphate buffer (pH 7.4) 258 overnight at 4°C. A subset of metacyclic parasites were fixed using an imidazole-based 259 technique to stain the neutral lipids [20] prior to fixation. All cells were washed using 260 the 0.1 M phosphate buffer (pH 7.4) and embedded in molten 2% agar (Merck). Agar 261 pellets containing the cells were post-fixed in a mixture of 1% phosphate-buffered 87 262 osmium tetroxide and 1.5% potassium ferrocyanide (final concentration) for 1 h and 263 processed for resin embedding (PolyBed 812, Polysciences, Warrington, PA). The 264 sections were mounted on uncoated 200-mesh copper grids and were viewed using a 265 transmission electron microscope (JEOL JEM-1230, Tachikawa, Tokyo). Grids were 266 examined at 50–120,000X magnification. 267 268 Immunogold Electron Microscopy 269 The infected macrophages and metacyclic L. i. chagasi were processed for immunogold 270 staining. Cells were fixed in 4% paraformaldehyde, 1% glutaraldehyde (Sigma, grade I), 271 and 0.02% picric acid in 0.1 M cacodilate buffer (pH 7.2) at 4 °C. Free aldehyde groups 272 were quenched in a 0.1-M glycine solution for 60 min. Cells were then dehydrated in a 273 methanol series and embedded at progressively lowered temperatures in Lowicryl K4M. 274 Thin sections containing the parasites were stained with mouse anti-PGFS antibody 275 (1:20), and the thin sections containing the infected macrophages were stained with 276 rabbit anti-FP receptor antibody (1:20) overnight at 4ºC. After incubation the sections 277 were washed with HBSS-/- and incubated with 10 ηm colloidal gold-AffiniPure- 278 conjugated anti-mouse or anti-rabbit IgG (H + L) for 1h at room temperature. The 279 samples were examined at 120,000X magnification using a transmission electron 280 microscope (JEOL JEM-1230, Tachikawa, Tokyo). 281 282 Statistical Analyses 283 Each experiment was repeated at least three times. The data are presented as the mean 284 plus SEM (standard error) of representative experiments and were analyzed using the 285 GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). The dose- 286 response experiments were analyzed using one-way ANOVA with post-test to linear 88 287 trend, and comparisons between the two groups were analyzed using Student´s t-test. 288 The differences were considered statistically significant when p ≤ 0.05. 289 290 Results 291 Lipid Body Arrangement During L. i. chagasi Metacyclogenesis 292 Lipid bodies can be visualized using techniques to stain neutral lipids, such as osmium 293 impregnation or BODIPY (a fluorescent probe) [21]. Both light and confocal 294 microscopic analyses were used to visualize and enumerate the LBs content in the 295 different developmental forms of L. i. chagasi (Figure 1A-F). We used the LcJ L. i. 296 chagasi parasite cell line that converts between promastigote and amastigote forms in 297 axenic culture [18]. Graphical representation of the numbers of lipid bodies per parasite 298 showed that LcJ amastigotes contained more LBs per cell than LcJ promastigotes in 299 logarithmic stage growth (Figure 1G). Similarly, wild-type (wt) L. i. chagasi 300 amastigotes isolated from spleens of infected hamsters contained more LBs than 301 promastigotes (Figure 1G). Remarkably, the LB content increased during 302 metacyclogenesis, with the lowest numbers in logarithmic, higher in unpurified 303 stationary and highest content in isolated metacyclic forms. The amount of LBs per 304 metacyclic promastigote cell did not differ statistically from LB number per amastigote 305 (Figure 1G). Next we investigated the ultrastructural arrangement of LBs in the 306 metacyclic forms of the L. i. chagasi, because this is the infective stage of the parasite. 307 Confocal microscopy showed that the LBs were arranged in a linear sequence near to 308 the cell nucleus (Figure 2A-B). In addition, we confirmed that the observed structures 309 were LBs using osmium imidazole-based (Figure 2C) and conventional Transmission 310 Electron Microscopy (TEM) (Figure 2D). The TEM analysis clearly showed the 89 311 location of the LBs close to the mitochondrion and cell nucleus in the metacyclic forms 312 (Figure 2D). 313 314 L. i. chagasi Lipid Bodies are Intracellular Sites for the Production of PGF2α 315 In Trypasomatidae, the only two enzymes in the eicosanoid synthesis pathway that have 316 been described are phospholipase A2 and PGFS [14]. To address whether PGFS is 317 associated with LBs and whether this association correlates with virulence of parasite 318 forms, we generated an anti-PGFS mouse antiserum against L. chagasi PGFS 319 recombinant protein (Supporting Information Figure S1A-B). The LcJ promastigotes 320 expressed higher levels of PGFS than amastigotes (Figure 3A). Strikingly, the PGFS 321 expression in L. i. chagasi metcyclic forms was increased compared to wt amastigotes 322 and procyclic forms (Figure 3A). 323 Lipid bodies are intracellular sites of eicosanoid synthesis in mammalian cells [22]. We 324 tested if this is the case for L. i. chagasi by investigating the subcellular localization of 325 PGFS in the metacyclic forms of the parasite. We verified that staining for PGFS was 326 strictly localized in the LBs (Figure 3B-D). Furthermore, we incubated wt L. i. chagasi 327 procyclic forms with different doses of arachdonic acid (AA) (3.75 – 30 µM), a major 328 eicosanoid precursor. AA induced both LB formation and a dose-dependent release of 329 PGF2α by Leishmania (Figure 4B-C). However, there was no detectable effect on the 330 cellular content of PGFS in the AA-stimulated L. i. chagasi procyclic forms (Figure 331 4A). These results suggest that: (i) L. i. chagasi LBs are the intracellular sites for PGF2α 332 production, (ii) the promastigote production of PGF2α increases in response to AA and 333 (iii) this prostaglandin is released from the parasite to the extracellular environment. 334 Because compartmentalization is an important component of eicosanoid synthesis, a 90 335 failure to induce the total cellular abundance of the PGFS biosynthetic enzyme does not 336 signify a failure to increase its activity. 337 338 Leishmania-driven PGF2α promotes L. i. chagasi infection of macrophages 339 Several intracellular pathogens induce LB formation and recruitment to parasitophorous 340 vacuoles [22]. Intriguingly, our data suggest that the different developmental stages of 341 L. i. chagasi forms did not induce host cell LB formation during infection of bone 342 marrow-derived macrophages (BMMs). In contrast, we observed that the LB staining 343 was restricted to the L. i. chagasi cell itself within the infected BMM (Figure 5A-B; 344 Figure 7A-D and Video S1). Because we have documented PGF2α release from L. i. 345 chagasi, we decided to assess the role of this eicosanoid in BMM infection. PGF2α acts 346 directly on the FP receptor and triggers the activation of the COX pathway [15]. The 347 distribution of FP receptor was observed by confocal immunofluorescence in uninfected 348 and L.i. chagasi -infected BMMs for 1h (Figure 6). The FP receptor staining in 349 uninfected cell present diffuse in the cytoplasm while in infected cell it was punctual 350 and near to the early phagocytic vacuoles and to parasitophorous vacuoles containing 351 parasites (Figure 6). Using immunogold TEM to investigate BMMs infected for 1 hr 352 with L.i. chagasi, we observed that the FP receptor, which recognizes PGF2α, was 353 localized near to the parasitophorous vacuoles (Figure 7E-F). 354 In addition, BMMs triggered a rapid expression of COX-2 mRNA after 1-4 hours of 355 infection (Figure 8A). Surprisingly, the infected BMMs did not release PGF2α at the 356 early time points (Figure 8B). These results suggest that the COX-2 products of AA 357 metabolism could be being internalized and used by the parasites. Accordingly, 358 pretreatment of BMMs with AL8810, a specific inhibitor of the FP receptor, resulted in 359 a dose-dependent decrease in L. i. chagasi infection (Figure 9A-C). Furthermore, 91 360 inhibition of the FP receptor decreased the infection index levels in BMMs infected 361 with all forms of parasites examined, i.e., amastigotes, procyclics and metacyclics 362 (Figure 9A-C). Taken together, these results indicate that PGF2α plays an important role 363 in L. i. chagasi infection. 364 365 Discussion 366 Lipid bodies can play important roles as nutritional sources and in eicosanoid 367 production during host-pathogens interactions [23,24]. Eicosanoids released by 368 macrophage LBs have the potential to modulate immune response [10,22]. Despite of 369 this, the role of eicosanoids produced by parasites and the cellular mechanism involved 370 in their production have not been previously addressed. In the present study, we 371 demonstrate that the LBs in L. i. chagasi are intracellular sites of prostaglandin 372 production. Because LBs increase during both metacyclogenesis and in the intracellular 373 amastigote form, we hypothesize that they could act as virulence factors. In addition, the 374 LBs in L .i. chagasi are responsible for the production of PGF2α, which we also 375 demonstrate here that is important for the modulation of macrophage infection. 376 LBs have been associated with other infectious agents, such as T. gondii and P. 377 falciparum [10]. The increase in the number of LBs in these parasites was demonstrated 378 in in vitro cultures and is associated with the acquisition of lipids, such as 379 triacylglycerol (TAG), from the host cell during infection [25]. Herein, we demonstrate 380 that L. i. chagasi increases the lipid storage in the LBs and amplifies the expression of 381 PGFS during metacyclogenesis, demonstrating that the parasites can mobilize the 382 eicosanoid machinery in the infective forms of the parasite. 383 The biology of LBs in mammalian cells is relatively well understood. In leukocytes, LB 384 formation is a coordinated process involving the activation of receptors and kinase 92 385 proteins [2]. Similarly, recent studies in leukocytes have shown that T. brucei modulate 386 the LB number via the activation of a specific parasite kinase named lipid droplet kinase 387 LDK [26]. In the current study, we found that AA, a substrate of parasite PGFS, 388 increases both the number of LBs and the release of PGF2α by L. i. chagasi. Previous 389 studies have shown that AA induce parasites to release prostaglandins, such as PGE2, 390 PGD2 and PGF2α [13,14,27,28]. Here we extend these observations and show an 391 association of these mediators with parasite infectivity. Our data suggest that L. i. 392 chagasi-derived PGF2α may be important for parasite virulence because the expression 393 of PGFS in the parasite increase during metacyclogenesis. In addition, the PGFS is 394 expressed predominantly in LBs, indicating that LBs are the major intracellular site for 395 the production of prostaglandins in L. i. chagasi (Figure 10). 396 It has been reported that the host cell LBs are an important source of TAG and 397 cholesterol for pathogens [23]. Indeed, pathogens can recruit host cell LBs to their 398 parasitophorous vacuoles during infection [3,6]. A recent study suggested that 399 Leishmania may use a similar mechanism to acquire lipids and to induce foam cell 400 formation [29]. However, our data demonstrated that the LBs formed during the L. i. 401 chagasi infection are exclusively from the parasites because the LBs are located inside 402 the parasites within the parasitophorous vacuoles in the infected macrophages. Further 403 studies will be necessary to elucidate how Leishmania acquires lipids from the host cells 404 for its metabolism. 405 The role of PGF2α in the immune response is not well understood. Macrophages can 406 produce PGF2α during inflammation [30] or during L. donovani infection [27]. PGF2α 407 ligates and activates the FP receptor to enhance COX-2 expression in the 3T3-L1 cell 408 line, and the autocrine signaling of this mediator increases PGE2 and PGF2α levels [15]. 409 Herein, we demonstrate that the FP receptor is localized in the early phagocytic 93 410 vacuoles and surface parasitophorous vacuoles during macrophages infection with 411 metacyclic forms of L. i. chagasi (Figure 10). In addition, the L. i. chagasi-infected 412 macrophages rapidly express COX-2 but do not release PGF2α. These results are 413 consistent with previous studies showing that Leishmania infections trigger COX-2 414 expression [29,31–33]. We hypothesize that the COX-2 expression observed in the L. i. 415 chagasi-infected macrophages is induced by the PGF2α released from parasites, and that 416 the metabolites from COX-2 enzyme, such as prostaglandin H2 (PGH2), in the 417 macrophages could be harvested by the L. i. chagasi inside the parasitophorous 418 vacuoles. We further reinforce this idea by showing that the inhibition of the FP 419 receptor in the macrophages diminishes the L. i. chagasi parasite load 72h after 420 infection. 421 Our findings demonstrate that LBs and PGFS from L. i. chagasi are upregulated in the 422 metaclyclic forms of the parasites and the role of PGF2α and your FP receptor in the 423 Leishmania-host interplay. They also suggest that parasite derived eicosanoids may 424 enhance the survival of the parasite inside macrophages. Further studies will be 425 necessary to elucidate how intracellular Leishmania could acquire lipids from the host 426 cells and if and how they in turn release eicosanoid precursors into the infected 427 macrophage cytoplasm. Ultimately this could reveal a major mechanism through which 428 the parasite controls the inflammatory microbicidal state of the infected host cell. 429 430 431 Acknowledgments 432 We thank Dr. Bruno Bezerril Andrade, Dr. Petter F. Entringer, Msc. Leonardo Arruda, 433 Dr. Claudia Ida Brodskyn and Dr. Marcelo T. Bozza for their helpful discussions, and 434 Dr. Adriana Rangel and Dr. Claudio Figueira for their technical assistance with the 94 435 TEM and Dr Jian Shao, Dr Milena Soares and Carine M. Azevedo for his assistance 436 with confocal microscopy. We also thank to Dr. Jason L. Weirather and Dr. Bradjsh 437 Kumar Singh for their technical help in the laboratory and for their discussions. 438 439 References 440 441 1. 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(D-F) 547 show merged images of confocal microscopy of the parasites to LBs stained with 548 BODIPY (green), DNA stained with ethidium bromide (red), and cell contours (DIC). 549 (G) shows the number of LBs in the different stages of Leishmania including the 550 amastigote and promastigote LcJ axenic parasites strain and wt parasites. Ama: 551 Amastigote; Pro: Procyclic; Log: Logarithmic; Sta: Stationary: Meta: Metacyclic. Bars 552 represent the mean ± SEM from LB per parasite; n = 3; ***, p<0.001 between groups 553 (One-way ANOVA). 554 555 Figure 2. Cellular characterization of LBs in metacyclic promastigotes of L. i. 556 chagasi. (A) Schematic representation of the arrangement of the LBs in most 557 metacyclic forms, also shown microscopically in (B) by merge between LBs (green), 558 DNA (red), and cell contours (DIC). Neutral lipids were detected using osmium 559 imidazole-based (C) or conventional TEM (D). Lipid bodies are indicated with white 560 arrowheads. C-D, Left panels show details of indicated LBs. m – mitochondrion; k – 561 kinetoplast. 562 563 Figure 3. Leishmania LBs are intracellular sites for the production of PGF2α. (A) 564 Immunoblot comparing the abundance of PGFS at different stages in the wild-type (wt) 565 or LcJ strain of L. i. chagasi, as described in the methods section. Blots were incubated 566 with polyclonal antiserum to recombinant PGFS (see Figure S1A- B). B, left panel 567 shows merged image of metacyclic promastigotes visualized by confocal microscopy 98 568 with anti-PGFS (blue), LBs stained with BODIPY (green), DNA stained with ethidium 569 bromide(red), and cell contours (DIC). B, right panels show the left white box area as 570 individual stains and a merged image to visualize PGFS co-localization with LBs. C and 571 D show PGFS localized close to the LBs in the metacyclic forms of two different 572 parasites by post-embedding immunogold staining (120k-fold increase). Black 573 arrowheads indicate the immunogold staining of PGFS. 574 575 Figure 4. LBs formation and PGF2α release are modulated by arachdonic acid. (A) 576 Immunoblot documents the abundance of PGFS in the procyclic forms of L. i. chagasi 577 stimulated with arachdonic acid (AA), vehicle (veh) or buffer (CTR) for 12 h. (B) 578 Parasites were incubated with different doses of AA (3.75 – 30 µM) for 72 h, and then 579 stained with osmium tetroxide to enumerate the LBs. (C) Supernatants from 580 promastigotes in panel B were harvested and PGF2α levels were measured. Significance 581 was tested by one-way ANOVA with post-test linear trend. Bars represent the mean ± 582 SEM, n = 3. 583 584 Figure 5. LBs are restricted to parasites during macrophage infection. (A) shows 585 images of BMMs infected with LcJ amastigotes and promastigotes for 24 h. Nuclei 586 were stained with ethidium bromide (red), parasitophorous vacuole (PV) membranes 587 were stained with anti-Lamp1 (blue), and LBs were stained with BODIPY (green). (B) 588 shows a z-section sequence of images through an infected BMM. White arrowheads 589 indicate the LBs inside PVs after 1 hour of LcJ amastigote infection (see Video S1). 590 591 Figure 6. Localization of FP receptor during early macrophage infection. BMMs 592 were infected or not with metacyclic L. i. chagasi for 1h and FP receptor localization 99 593 was shown in the uninfected (left panel) and infected cells (right panels). Nuclei were 594 stained with DAPI (red), parasitophorous vacuole (PV) membranes were stained with 595 anti-Lamp1 (blue), and PGF2α receptors (FP) were stained using anti-FP receptor or IgG 596 control (green). Merge of fluorescence and differential interference contrast (DIC) 597 microscopy shows images from uninfected and infected. 598 599 Figure 7. LBs and FP receptor arrangement in the Leishmania-infected 600 macrophage. Transmission electron microscopic images of BMMs after 1h infection 601 with metacyclic L. chagasi are shown. (A) shows an infected BMM with 602 parasitophorous vacuoles (PV) outlined in white. Panels B (80k-fold increase), C, and 603 D (120k-fold increase) show details of LBs inside the parasites. (E) shows post- 604 embedding immunogold staining for FP receptor (50k-fold increase). (F) shows details 605 of FP receptor arrangement close to the PVs in the black box region from the panel B 606 (120k-fold increase). FP receptor staining is indicated by black arrowheads. P – 607 parasite. 608 Figure 8. COX-2 expression and PGF2α release during macrophage infection. (A) 609 shows the COX-2 transcript levels measured using qPCR in BMMs infected with LcJ 610 amastigotes, promastigotes and wt metacyclics for 1, 4 and 24 hours and processed 611 immediately. * p<0.05 (Student’s t-test). (B) shows the kinetic of PGF2α levels released 612 by BMMs infected with L. i. chagasi metacyclic forms for 1-48 hours. * p<0.05 613 (Student’s t-test). 614 615 Figure 9. Inhibition of the FP receptor hampers L. i. chagasi infection. BMMs were 616 pretreated for 1 h with AL8810 (50-1 µM), a FP receptor antagonist, and infected with 617 (A) LcJ amastigotes, (B) promastigotes or (C) wt metacyclic forms of the parasite for 72 100 618 h. Infection index is illustrated (One-way ANOVA with post-test´s linear trend). Bars 619 represent the mean ± SEM, n = 3. 620 621 Figure 10. Schematic view of LB formation and PGF2α release in L. i. chagasi 622 during macrophage infection. (i) LBs are intracellular sites of PGF2α in L. i. chagasi. 623 PGFS is localized in the LBS and increase during metacyclogenesis. In addition, LBs 624 and PGF2α can be up regulated by AA in promastigote forms. (ii) FP receptor is 625 mobilized to macrophages PVs, and there it is activated by PGF2α increasing parasite 626 infectivity. 627 628 Supporting Information 629 630 Figure S1. Specificity of antiserum against prostaglandin F synthase from L. i. chagasi. 631 (A) C57BL/6 mice were immunized intraperitoneally with three doses of PGFs 632 recombinant protein (30 µg) plus incomplete Freud´s adjuvant (IFA), and the serum 633 conversion was measured using ELISA using plates coated with recombinant PGFS. (B) 634 Immunoblot showing the specific binding of the PGFS antiserum in to membranes 635 containing L. chagasi total promastigote lysate. 636 637 Video S1. LBs are restricted to parasitophorous vacuoles during macrophage infection. 638 BMMs were infected with LcJ amastigotes at an MOI of 3 parasites:1 macrophage for 1 639 h. Nuclei were stained with ethidium bromide (red), parasitophorous vacuole (PV) 640 membranes were stained with anti-Lamp1 (blue), and LBs were stained with BODIPY 641 (green). The movie shows the z-section sequence of images. 642 101 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 Figure 1 102 668 669 670 671 672 673 674 675 676 677 678 679 680 681 Figure 2 103 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 Figure 3 104 707 Figure 4 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 Figure 5 105 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 Figure 6 106 757 Figure 7 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 Figure 8 107 782 Figure 9 783 784 785 786 787 788 789 790 Figure 10 791 792 793 794 795 796 797 798 799 800 801 802 803 804 Figure S1 108 5. DISCUSSÃO Sob condições inflamatórias, eicosanoides são prioritariamente produzidos em organelas citoplasmáticas denominadas corpúsculos lipídicos, os quais são formados em leucócitos e outras células envolvidas na resposta inflamatória às infecções e diversos outros estímulos (BOZZA et al., 2009). Os eicosanoides exercem um importante papel na infecção por Leishmania. Nessa tese foram abordadas as participações de eicosanoides e corpúsculos lipídicos na interface da interação parasita-vetor-célula hospedeira. Nós verificamos que: (1) a saliva de L. longipalpis é capaz de modular a biogênese dos corpúsculos lipídicos e a produção de eicosanoides; (2) o perfil de mediadores lipídicos favorece o estabelecimento da infecção e possivelmente a transmissão do parasito e, além disso, (3) nós demonstramos os mecanismos pelo qual a L. i. chagasi produz eicosanoides e que estes também são importantes para a infectividade da forma metacíclica, a forma envolvida na fase inicial de transmissão do parasita do flebótomo para o hospedeiro vertebrado. A saliva de flebotomíneos induz uma resposta inflamatória caracterizada pelo influxo celular seguido por um mecanismo de supressão da resposta imunológica e hemostática do hospedeiro (ANDRADE et al., 2005). Nosso grupo de pesquisa e outros tem demonstrado o papel da saliva como marcador epidemiológico e como modulador da resposta imune do hospedeiro (CHARMOY et al., 2010; PETERS; SACKS, 2009) (MANUSCRITO II). Entretanto, a participação da saliva na indução de eicosanoides, bem como sua associação com a biogênese de corpúsculos lipídicos ainda não haviam sido investigadas até o presente estudo. Aqui, nós mostramos que a saliva de L. longipalpis induz a formação de corpúsculos lipídicos e produção de PGE2 em macrófagos peritoneais ex vivo e in vitro via a fosforilação de quinases e ativação de COX-2 (MANUSCRITO I). 109 Estudos anteriores demonstraram em vários modelos experimentais que a saliva de flebótomo é capaz de induzir o recrutamento celular (CARREGARO et al., 2008; MONTEIRO et al., 2007; SILVA et al., 2005; TEIXEIRA et al., 2005). Peters e cols. (2008) mostraram um perfil semelhante de recrutamento durante a picada de flebótomo usando um sistema de aquisição de imagem intravital. Aqui, nós confirmamos os relatos anteriores de que a saliva de L. longipalpis induz um infiltrado inflamatório composto principalmente de macrófagos e neutrófilos. Além disso, mostramos que o recrutamento celular induzido pela saliva ocorre concomitante com a produção de PGE2 e LTB4 (MANUSCRITOS I e III). Neste cenário, os eicosanoides poderiam estar deflagrando o recrutamento celular. A produção de LTB4 por macrófagos residentes é responsável por induzir a migração de neutrófilos (OLIVEIRA et al., 2008). Além disso, outros estímulos inflamatórios como o LPS induzem a migração de macrófagos através da produção de PGD2 e PGE2 (TAJIMA et al., 2008). A PGE2 é o eicosanoide mais comumente produzido por células inflamatórias, e que é conhecido por exercer efeitos anti-inflamatórios e vasodilatadores. Esses efeitos são úteis para a manutenção da hematofagia de alguns insetos. A saliva do carrapato Ixodes scapularis, por exemplo, contém níveis farmacológicos de PGE2, o qual está implicado na atividade imunomoduladora da saliva na ativação de células dendríticas e macrófagos (SÁ-NUNES et al., 2007). Estudos anteriores utilizando a saliva de Phlebotomus sugerem que as propriedades anti-inflamatórias da saliva podem ser atribuídas à produção PGE2 e IL-10 por células dendríticas (CARREGARO et al., 2008; MONTEIRO et al., 2005). Nestes estudos, o recrutamento celular induzido pela estimulação OVA foi inibido em presença da saliva, o qual foi associado com um perfil anti-inflamatório dependente da produção de IL-10, IL-4 (MONTEIRO et al., 2005) e PGE2 (CARREGARO et al., 2008). Já a saliva de L. longipalpis contém o maxadilan, 110 um peptídeo vasodilatador com atividades imunomoduladoras que é capaz de induzir em macrófagos ativados com LPS a produção de PGE2 via ativação de COX-1 (SOARES et al., 1998). Aqui, nós demonstramos que a saliva de L. longipalpis induz a produção de PGE2 em macrófagos residentes pela ativação da COX-2, uma vez que a inibição farmacológica com NS-398 reverteu esse efeito da saliva (MANUSCRITO I). Além disso, nós investigamos a presença de PGE2 na saliva de L. longipalpis, mas não encontramos níveis detectáveis deste eicosanoide (dado não mostrado). Corpúsculos lipídicos de células inflamatórias podem conter enzimas relacionadas com o metabolismo de eicosanoides tais como a COX e 5-LO (BOZZA et al., 2009). Estudos anteriores têm mostrado que vários estímulos inflamatórios e infecciosos são capazes de induzir a formação de CLs em macrófagos (BOZZA; MELO; BANDEIRA-MELO, 2007; BOZZA et al., 2009). Nós verificamos que a saliva L. longipalpis induz a formação de CLs em macrófagos in vivo e in vitro, sugerindo que a saliva atua diretamente sobre estas células. Além disso, os CLs induzidos em macrófagos pela saliva de L. longipalpis parecem estar comprometidos com a produção de PGE2, uma vez que nós observamos a co-localização das enzimas COX-2 e PGEsintase nestas organelas (MANUSCRITO I). Dados referentes ao efeito direto dos componentes da saliva de L. longipalpis sobre vias de sinalização nas células hospedeiras são escassos. MAP quinases como ERKs e proteína quinase C (PKC), estão entre as principais enzimas envolvidas na sinalização nas respostas celulares, incluindo a produção de eicosanoides. As quinases ERK1 e ERK2 induzem a ativação de cPLA2, uma enzima que hidrolisa fosfolipídios de membrana liberando o AA, o qual é metabolizado em prostaglandina H2 pelas COXs (BOZZA et al., 2009). Estudos anteriores demonstraram a compartimentalização em CLs de MAP quinases e cPLA2 (MOREIRA et al., 2009; YU et al., 1998), bem como de 111 COX-2 e PGE-sintase (ACCIOLY et al., 2008; D’AVILA et al., 2006; PACHECO et al., 2002). Aqui, nós verificamos que a saliva de L. longipalpis ativa a fosforilação de ERK-1/2 e PKC-α em macrófagos (MANUNSCRITO I). A ativação de COX-2 e a produção de PGE2 em macrófagos estimulados com LPS são dependentes da fosforilação de quinases tais como PKC-α (GIROUX; DESCOTEAUX, 2000) e ERK-1/2 (WEST et al., 2000). Nós mostramos que a produção de PGE2 induzida pela saliva de L. longipalpis é dependente da atividade de ERK-1/2 e PKC-α (MANUSCRITO I). Esta associação entre a ativação de quinases e o metabolismo de eicosanoides dentro de CLs pode servir para aumentar a rápida produção de eicosanoides em resposta a estímulos extracelulares tais como a saliva. Além do seu papel na regulação da resposta do hospedeiro à infecção pela modulação da produção de eicosanoides, os CLs também podem servir como fontes ricas de nutrientes para os patógenos intracelulares, favorecendo assim a replicação intracelular patógeno (BOZZA et al., 2009; D’AVILA; MAYA-MONTEIRO; BOZZA, 2008). Apesar de grande parte dos estudos realizados sobre eicosanoides na infecção por Leishmania envolver espécies que acometem o sistema tegumentar, parece claro que existe uma dicotomia na resposta imune, em que a produção de produção de PGE2 beneficia a viabilidade do parasita (AFONSO et al., 2008; LONARDONI et al., 1994; PINHEIRO et al., 2008), enquanto que a produção de LTB4 favorece a resolução da infecção (SEREZANI et al., 2006). Por outro lado, Ansted e cols. (2001) demonstraram de forma elegante que a produção de PGE2 facilitava a visceralização de L. donovani em animais submetidos a uma dieta com restrição de Cu e Zn, mas não afetava a parasitemia dos animais infectados (Ansted et al.; 2001), sugerindo que em outras espécies de Leishmania o efeito da PGE2 poderia estar associado a disseminação do parasita. A maioria dos estudos envolvendo eicosanoides negligencia em quais etapas 112 da infecção os eicosanoides poderiam estar envolvidos. Aqui, nós mostramos que a saliva modula o perfil de eicosanoides de maneira que a ativação de COX-2 coordena a produção de PGE2 em detrimento da produção de LTB4 nos momentos iniciais da infecção por L. i. chagasi (MANUSCRITO III). A importância da produção de PGE2 para o estabelecimento da infecção foi demonstrada para alguns patógenos (D’AVILA; MAYA-MONTEIRO; BOZZA, 2008). Em ratos e camundongos, a infecção com Trypanosoma cruzi induz produção de PGE2 por macrófagos (D’AVILA et al., 2011; FREIRE-DE-LIMA et al., 2000; MELO et al., 2003). Um dos fatores responsáveis pela indução da produção de PGE2 por macrófagos é o reconhecimento de células apoptóticas (FREIRE-DE-LIMA et al., 2000). A interação entre neutrófilos apoptóticos e macrófagos aumenta a infecção por Mycobacterium bovis via o aumento dos níveis de PGE2 e TGF-β1 (D’AVILA et al., 2006). Um mecanismo similar foi demonstrado para infecção por L. amazonensis, onde a interação entre neutrófilos apoptóticos e macrófagos humanos aumentou a infecção com a participação de PGE2 e TGF-β1 (AFONSO et al., 2008). A saliva de L. longipalpis aumenta a apoptose de neutrófilos ao mesmo tempo em que aumenta a produção de PGE2 durante a infecção por L. i. chagasi in vitro (PRATES et al., 2011). In vivo, é possível notar a interação entre macrófagos e neutrófilos infectados, após poucas horas da infecção por L. i. chagasi (dado não mostrado). Aqui, nós observamos que a saliva de L. longipalpis reduz a produção de LTB4 nos momentos iniciais da infecção por L. i. chagasi, ao mesmo tempo que estimula uma resposta anti-inflamatória pelo aumento da produção de PGE2 (MANUSCRITO III). Este ambiente induzido pela saliva em que prevalece a produção de PGE2 sobre LTB4 aumenta a viabilidade dos parasitas dentro das células peritoneais. Neste sentido, nós verificamos que a inibição farmacológica de COX-2 reverteu o efeito 113 da saliva de L. longipalpis sobre a viabilidade dos parasitas (MANUSCRITO III), sugerindo que a presença da saliva favorece um balanço inflamatório que poderia facilitar a transmissibilidade e infecção de L. i. chagasi , uma vez que eicosanoides podem ser produzidos mais rápido do que outros mediadores tais como citocinas e quimiocinas, os quais precisam ser expressos de novo. A despeito da produção de eicosanoides pela célula hospedeira, parasitas também são capazes de produzir eicosanoides (KUBATA et al., 2007). Entretanto, o mecanismo celular envolvido nesta produção, bem como a importância dos eicosanoides produzidos pelo parasito para a infecção permanece por ser esclarecida. Nós demonstramos que os CLs de L. i. chagasi são sítios intracelulares de produção de prostaglandina (MANUSCRITO IV). Uma vez que os CLs de L. i. chagasi aumentam em número durante a metaciclogênese nós acreditamos que os CLs e as PGs proveniente destes CLs sejam fatores de virulência em L, i. chagasi (MANUSCRITO IV). Os corpúsculos lipídicos têm sido associados com a virulência de diversos patógenos, tais como T. gondii e P. falciparum (SAKA; VALDIVIA, 2012). O aumento no número de CLs nos parasitas foi demonstrado em culturas in vitro e está associado com a aquisição de lipídeos como o triacilglicerol (TAG) da célula hospedeira durante a infecção por Toxoplasama (NISHIKAWA et al., 2005). Aqui, nós demonstramos que L. i. chagasi aumenta o estoque de lipídios em CLs durante a metaciclogênese (MANUSCRITO IV), sugerindo que os parasitas podem mobilizar o metabolismo lipídico em suas formas infectivas. A biologia dos CLs de leucócitos e outras células de mamíferos é relativamente bem conhecida. Em leucócitos, a formação de CLs é um processo controlado e que envolve a ativação de receptores de membrana, a fosforilação de proteínas quinase e a 114 produção de eicosanoides (BOZZA; MAGALHÃES; WELLER, 2009). Similarmente, um estudo recente mostrou que a formação de CLs em T. brucei depende da ativação de uma quinase específica do parasita denominada proteína quinase de corpúsculo lipídico (LDK) (FLASPOHLER et al., 2010). Entretanto a associação dos CLs de outras células eucarióticas que não as mamíferas, ainda não haviam sido associadas à produção de eicosanoides até o presente estudo. Leishmania não possui PLA2 descrita em seu genoma e não apresenta proteínas análogas às COXs para o metabolismo de AA à eicosanoides. Kabutu e cols. (2003) descreveram a presença de uma PGFS em L. donovani capaz de metabolizar AA à PGF2α (KABUTUTU et al., 2003). Aqui, nós verificamos que a expressão da PGFS de L. i. chagasi aumenta durante a metaciclogênese. Além disso, a PGFS foi localizada predominantemente em CLs, indicando que CLs são os principais sítios intracelulares para a produção de prostaglandinas em L. i. chagasi (MANUSCRITO IV), sugerindo que este pode ser um fator de virulência. A quantidade de CLs e a produção de eicosanoides podem ser moduladas pela presença de AA (BOZZA et al., 2002; MOREIRA et al., 2009; WELLER; DVORAK, 1985). Estudos anteriores mostraram que o tratamento com AA induz L. donovani a produzir as prostaglandinas PGE2, PGD2 e PGF2α (KABUTUTU et al., 2003; KUBATA et al., 2000, 2007). Nós estendemos esses achados e demonstramos que a incubação de L. i. chagasi com AA aumenta tanto a quantidade de CLs, quanto a produção de PGF2α, embora a expressão da PGFS permaneça quase inalterada (MANUSCRITO IV). Corpúsculos lipídicos das células hospedeiras são importantes fontes de TAG e colesterol para os patógenos (MURPHY, 2012). Além disso, patógenos podem recrutar CLs das células hospedeiras para o vacúolo parasitóforo durante a infecção (COCCHIARO et al., 2008; D’AVILA et al., 2011). Um estudo recente sugeriu que 115 Leishmania pode utilizar um mecanismo similar para aquisição de lipídios do hospedeiro (RABHI et al., 2012). Entretanto, nossos dados sugerem que os CLs formados durante a infecção são exclusivamente do parasito intracelular, uma vez que os CLs estão restritos aos parasitas dentro dos vacúolos parasitóforos dos macrófagos infectados (MANUSCRITO IV). Estudos posteriores serão essenciais para elucidar como Leishmania adquire lipídios da célula hospedeira para o seu metabolismo. O papel do PGF2α na resposta imune ainda não havia sido elucidado até o presente estudo. Macrófagos produzem PGF2α durante a inflamação (LEE et al., 2012) ou durante a infecção por L. donovani (REINER; MALEMUD, 1985). PGF2α se liga, ativa o receptor FP e induz a expressão de COX-2 em células de linhagem 3T3-L1, e a sinalização autócrina deste mediador aumenta a produção de PGE2 e PGF2α (UENO; FUJIMORI, 2011). Aqui, nós verificamos que o receptor FP está localizado na superfície dos vacúolos parasitóforos de L. i. chagasi nos momentos iniciais da infecção. Além disso, macrófagos infectados com L. i. chagasi expressaram rapidamente COX-2 mas não liberaram PGF2α (MANUSCRITO IV). Nossos resultados são consistentes com estudos anteriores que mostraram que a infecção com Leishmania ativa a expressão de COX-2 (GIROUX; DESCOTEAUX, 2000; GREGORY et al., 2008; MATTE et al., 2001). Nós hipotetizamos que a expressão de COX-2 observada em macrófagos infectados é induzida pelo PGF2α produzido pelos parasitas e que os metabólitos da enzima COX-2, tais como a prostaglandina H2 (PGH2) poderiam ser captados pela L. i. chagasi nos vacúolos parasitóforos (MANUSCRITO IV). Essa idéia é reforçada pela evidência encontrada durante a inibição do FP receptor em macrófagos, a qual reduziu a carga parasitária nos macrófagos infectados (MANUSCRITO IV). Esses dados sugerem que a PGF2α atua beneficiando a L. i. chagasi durante a infecção. 116 Em conjunto, os nossos dados sugerem que tanto o balanço de eicosanoides modulado pela saliva, quanto à prostaglandinas produzidas pela L. i. chagasi desempenham um papel importante nos momentos iniciais da infecção. Embora não tenha sido o foco desse estudo, nós nos perguntamos quais seriam as implicações dos nossos achados na LV crônica. Não existem dados experimentais ou clínicos sobre o status de produção dos eicosanoides durante a LV. Em uma análise preliminar nós verificamos que os níveis de PGE2 no soro de pacientes adultos com LV não alteram com a infecção, enquanto que os níveis de PGF2α estiveram aumentados em relação aos grupos de indivíduos assintomáticos (ver Anexo). Esses dados sugerem que PGF2α pode ser importante para a infecção por L. i. chagasi mesmo durante a fase crônica da doença. Estudos posteriores serão necessários para avaliar o papel das prostaglandinas durante a doença estabelecida e serão importantes para estabelecer novos perfis de tratamento em pacientes com LV. 117 6. CONCLUSÕES A saliva de L. longipalpis induz a formação de CLs em macrófagos associada a produção de PGE2 via fosforilação de PKC-α e ERK-1/2 e ativação de COX-2; A produção de PGE2 induzida pela saliva de L. longipalpis favorece a viabilidade intracelular de L. i. chagasi in vivo em neutrófilos e macrófagos; Corpúsculos lipídicos são sítios intracelulares de produção de PGs em L. i. chagasi; Prostaglandina F sintase é localizada em CLs e aumenta durante a metaciclogênese de L. i. chagasi; A formação de CLs e a produção de PGF2α pode ser modulada pela presença de AA em formas procíclicas de L. i. chagasi; A infeção por L. i. chagasi não induz a formação de CLs em macrófagos; O receptor FP é mobilizado para o VP de macrófagos e é importante para infectividade de L. i. chagasi. 118 7. REFERÊNCIAS BIBLIOGRÁFICAS Aqui estão listadas as referências utilizadas na introdução e discussão geral da tese. As referências citadas apenas nos manuscritos não estão listadas nesta seção. ABDELMOULA, M. 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A.; BARRAL, A.; BORGES, V. M.; BARRAL-NETTO, M. Heme impairs prostaglandin E2 and TGF-beta production by human mononuclear cells via Cu/Zn superoxide dismutase: insight into the pathogenesis of severe malaria. Journal of immunology (Baltimore, Md. : 1950), v. 185, n. 2, p. 1196-204, 15 jul. 2010. LUZ, N. F.; ANDRADE, B. B.; FEIJÓ, D. F.; ARAÚJO-SANTOS, T.; CARVALHO, G. Q.; ANDRADE, D.; ABÁNADES, D. R.; MELO, E. V.; SILVA, A. M.; BRODSKYN, C. I.; BARRAL-NETTO, M.; BARRAL, A.; SOARES, R. P.; ALMEIDA, R. P.; BOZZA, M. T.; BORGES, V. M. Heme Oxygenase-1 Promotes the Persistence of Leishmania chagasi Infection. The Journal of Immunology, v. 188, n. 9, p. 4460-7, 2012. PRATES, D. B.; ARAÚJO-SANTOS, T.; LUZ, N. F.; ANDRADE, B. B.; FRANÇACOSTA, J.; AFONSO, L.; CLARÊNCIO, J.;MIRANDA, J. C.; BOZZA, P. T.; DOSREIS, G. A.; BRODSKYN, C.; BARRAL-NETTO, M.; BORGES, V. M.; BARRAL, A. Lutzomyia longipalpis saliva drives apoptosis and enhances parasite burden in neutrophils. Journal of leukocyte biology, v. 90, n. 3, p. 575-82, set. 2011. SILVA, T. R. M.; PETERSEN, A. L. O. A.; SANTOS, T. A.; ALMEIDA, T. F.; FREITAS, L. A. R.; VERAS, P. S. T. Control of Mycobacterium fortuitum and Mycobacterium intracellulare infections with respect to distinct granuloma formations in livers of BALB/c mice. Memórias do Instituto Oswaldo Cruz, v. 105, n. 5, p. 6428, ago. 2010. 129 Article Lutzomyia longipalpis saliva drives apoptosis and enhances parasite burden in neutrophils Deboraci Brito Prates,*,† Théo Araújo-Santos,*,† Nı́vea Farias Luz,*,† Bruno B. Andrade,‡ Jaqueline França-Costa,*,† Lilian Afonso,*,† Jorge Clarêncio,* José Carlos Miranda,* Patrı́cia T. Bozza,§ George A. DosReis,ⱍⱍ Cláudia Brodskyn,*,¶ Manoel Barral-Netto,*,†,¶ Valéria de Matos Borges,*,¶,1,2 and Aldina Barral*,†,¶,1,2 *Centro de Pesquisa Gonçalo Moniz (CPqGM)-Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil; †Universidade Federal da Bahia, Salvador, Brazil; ‡Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Besthesda, Maryland, USA; §Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; ⱍⱍInstituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil; and ¶ Instituto Nacional de Ciência e Tecnologia de Investigação em Imunologia (iii-INCT), Salvador, Bahia, Brazil RECEIVED FEBRUARY 25, 2011; REVISED MAY 3, 2011; ACCEPTED MAY 24, 2011. DOI: 10.1189/jlb.0211105 ABSTRACT Neutrophils are considered the host’s first line of defense against infections and have been implicated in the immunopathogenesis of Leishmaniasis. Leishmania parasites are inoculated alongside vectors’ saliva, which is a rich source of pharmacologically active substances that interfere with host immune response. In the present study, we tested the hypothesis that salivary components from Lutzomyia longipalpis, an important vector of visceral Leishmaniasis, enhance neutrophil apoptosis. Murine inflammatory peritoneal neutrophils cultured in the presence of SGS presented increased surface expression of FasL and underwent caspase-dependent and FasL-mediated apoptosis. This proapoptosis effect of SGS on neutrophils was abrogated by pretreatment with protease as well as preincubation with antisaliva antibodies. Furthermore, in the presence of Leishmania chagasi, SGS also increased apoptosis on neutrophils and increased PGE2 release and decreased ROS production by neutrophils, while enhancing parasite viability inside these cells. The increased parasite burden was abrogated by treatment with z-VAD, a pan caspase inhibitor, and NS-398, a COX-2 inhibitor. In the presence of SGS, Leishmaniainfected neutrophils produced higher levels of MCP-1 and attracted a high number of macrophages by chemotaxis in vitro assays. Both of these events were abrogated by pretreatment of neutrophils with bindarit, an inhibitor of CCL2/MCP-1 expression. Taken together, our data support the hypothesis that vector salivary proteins trigger caspase-dependent and FasL-medi- Abbreviations: bindarit⫽2 methyl-2-1-(phenylmethyl)-1H-indazol-3yl[methoxy] propanoic acid, CNPq⫽Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico, CPqGM-FIOCRUZ⫽Centro de Pesquisa Gonçalo Moniz-Fundação Oswaldo Cruz, H2DCFDA⫽dihydrodichlorofluorescein diacetate, L⫽ligand, PS⫽phosphatidylserine, SGS⫽salivary gland sonicate 0741-5400/11/0090-575 © Society for Leukocyte Biology ated apoptosis, thereby favoring Leishmania survival inside neutrophils, which may represent an important mechanism for the establishment of Leishmania infection. J. Leukoc. Biol. 90: 575–582; 2011. Introduction Neutrophils play complex roles in infection. They provide an important link between innate and adaptive immunity during parasitic infections [1, 2] but also undergo apoptosis and are ingested by macrophages, thereby triggering secretion of antiinflammatory mediators [1, 3, 4]. At the onset of Leishmania infection, neutrophils establish a cross-talk with other cells in the development of an immune response [5], but the ultimate outcome is controversial, as protective [6 – 8] and deleterious [9 –12] effects to the host have been shown. Leishmania is transmitted by bites from sandflies looking for a blood meal. Tissue damage caused by sandfly probing [10] and sandfly saliva [13] is a potent stimulus for neutrophil recruitment, which results in a rapid migration and accumulation of neutrophils at the site of the vector’s bite [10, 12, 14]. Pharmacological properties of the saliva from sandflies are diverse [15, 16], and we have shown recently that saliva from Lutzomyia longipalpis, the main vector of Leishmania chagasi in Brazil, triggers important events of the innate immune response [17]. Despite the recognition of the importance of phlebotomine saliva and neutrophils in the initial steps of leishmanial infection, the direct role of saliva on the parasiteneutrophil interplay has not been addressed. Recent studies demonstrated the presence of Leishmaniainfected apoptotic neutrophils at the sandfly bite site [10]; 1. These senior authors contributed equally to this work. 2. Correspondence: Centro de Pesquisa Gonçalo Moniz (CPqGM)-Fundação Oswaldo Cruz (FIOCRUZ), Av. Waldemar Falcão, Candeal, Salvador, Bahia, Brazil. E-mail: [email protected]; [email protected] Volume 90, September 2011 Journal of Leukocyte Biology 575 130 however, a possible role of the sandfly saliva in this phenomenon remains unclear. Herein, we show an important FasL- and caspase-dependent apoptosis effect of Lu. longipalpis SGS upon neutrophils. In addition, the SGS-induced apoptosis favors L. chagasi survival inside neutrophils. These results represent the first evidence of direct effects of Lu. longipalpis SGS on host neutrophils and bring implications for the innate immune response to Leishmania infection. MATERIALS AND METHODS Mice and parasites Inbred male C57BL/6 mice, aged 6 – 8 weeks, were obtained from the animal facility of CPqGM-FIOCRUZ (Bahia, Brazil). This study was carried out in strict accordance with the recommendations of the International Guiding Principles for Biomedical Research Involving Animals. All experimental procedures were approved and conducted according to the Brazilian Committee on the Ethics of Animal Experiments of the FIOCRUZ (Permit Number: 027/2008). L. chagasi (MCAN/BR/89/BA262) promastigotes were cultured at 25°C in Schneider’s insect medium, supplemented with 20% inactive FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. Sandflies and preparation of salivary glands Adult phlebotomines from a Lu. longipalpis colony from Cavunge (Bahia, Brazil) were reared at the Laboratório de Imunoparasitologia/CPqGM/ FIOCRUZ, as described previously [16]. Salivary glands were dissected from 5- to 7-day-old Lu. longipalpis females under a stereoscopic microscope (Stemi 2000; Carl Zeiss, Jena, Germany) and stored in groups of 10 pairs in 10 l endotoxin-free PBS at –70°C. Immediately before use, glands were sonicated (Sonifier 450; Brason, Danbury, CT, USA) and centrifuged at 10,000 g for 4 min. Supernatants of SGS were used for experiments. The level of LPS contamination of SGS preparations was determined using a commercially available Limulus amoebocyte lysate chromogenic kit (QCL1000, Lonza Bioscience, Walkersville, MD, USA); negligible levels of endotoxin were found in the salivary gland supernatant. All experimental procedures used SGS in an amount equivalent to 0.5 pair of salivary glands/ group, representing ⬃0.7 g protein [18]. Reagents Anti-Gr-1-FITC, anti-mouse CD178L-PE (FasL; CD95L), PE hamster IgG isotype control (anti-TNP), CBA mouse inflammation kit, neutralizing antibody anti-mouse FasL, and hamster IgG isotype control were purchased from BD Biosciences (San Jose, CA, USA). Anti-mouse Ly-6G Alexa Fluor 647 was from BioLegend (San Diego, CA, USA). Annexin-V, PI (apoptosis detection kit), and z-VAD-FMK were from R&D Systems (Minneapolis, MN, USA). NS-398 and DMSO were from Cayman Chemical (Ann Arbor, MI, USA). Proteinase K was from Gibco, Invitrogen (Grand Island, NY, USA). RPMI-1640 medium and L-glutamine, penicillin, and streptomycin were from Invitrogen (Carlsbad, CA, USA). Schneider’s insect medium and etoposide (VP-16) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nutridoma-SP was from Roche (Indianapolis, In, USA), and thioglycolate was from Difco (Detroit, MI, USA). Bindarit was from Angelini Farmaceutici (Santa Palomba-Pomezia, Rome, Italy). Inflammatory neutrophils Peritoneal exudate neutrophils were obtained as described previously [19]. Briefly, C57BL/6 mice were i.p.-injected with aged 3% thioglycolate solution. Seven hours after injection, peritoneal lavage was performed using 10 ml RPMI-1640 medium supplemented with 1% Nutridoma-SP, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. To remove adherent cells, exudate cells were incubated at 37°C in 5% CO2 for 1 h in 576 Journal of Leukocyte Biology Volume 90, September 2011 250-ml flasks (Costar, Cambridge, MA, USA); cells on supernatants were then recovered and quantified in a hemocytometer by microscopy. Cell viability was ⬎95%, as determined by trypan blue exclusion (data not shown). Nonadherent cells were stained with anti-Gr-1 and Ly-6G to assess purity and were subsequently analyzed by flow cytometry using CellQuest software (BD Immunocytometry Systems, San Jose, CA, USA). Gr-1⫹ Ly-6G⫹ cells were routinely ⬎95% pure. Neutrophil apoptosis assay For cell cultures, neutrophils (5⫻105/well) were cultured in 200 l RPMI1640 medium, supplemented with 1% Nutridoma-SP, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin in 96-well plates (Nunc, Denmark) in the presence of different doses of Lu. longipalpis SGS (0.5, 1.0, and 2.0 pairs/well). In some experiments, etoposide (20 M) or LPS (100 ng/well) was used as a positive control. Three hours and 20 h after stimuli, neutrophil apoptosis was assessed by PS, exposed in the outer membrane leaflet through labeling with annexin-V-FITC by FACS analyses in combination with PI nuclear dye [19]. Annexin-V specificity was tested using Ca2⫹-free buffer; binding was not observed in this case. Morphological criteria for apoptosis, such as separation of nuclear lobes and darkly stained pyknotic nuclei, were also applied for quantification purposes using cytospin preparations stained by Diff-Quick under light microscopy [19]. Neutrophils were graded as apoptotic or nonapoptotic after examination of at least 200 cells/slide. To FasL-blocked assays, neutrophils were pretreated with a neutralizing antibody specific for FasL (10 g/mL) or an IgG isotype control (10 g/mL) for 30 min before use. In some experiments, SGS was preincubated with sandfly antisaliva serum (0.5 salivary gland pair plus 50 l serum preincubated for 1 h at 37°C) [20] or with proteinase K (10 mg/ml) at 65°C for 2 h and then for 5 min at 95°C for enzyme inactivation before use. Anti-sandfly saliva serum Hamster-derivated serum was obtained as described previously [20]. Briefly, hamsters (Mesocricetus auratus) were exposed to bites from 5- to 7-day-old female Lu. longipalpis. Animals were exposed three times to 50 sandflies every 15 days. Fifteen days after the last exposure, serum was collected and tested for IgG antisaliva detection by ELISA. Human neutrophil assay Human blood from healthy donors was obtained from Hemocentro do Estado da Bahia (Salvador, Brazil) after donors had given written, informed consent. This study was approved by the Research Ethics Committee of FIOCRUZ-Bahia. Human neutrophils were isolated by centrifugation using PMN medium, according to the manufacturer’s instructions (Robbins Scientific, Sunnyvale, CA, USA). Briefly, blood was centrifuged for 30 min at 300 g at room temperature. Neutrophils were collected and washed three times at room temperature by centrifugation at 200 g. Cells/well (106) were cultured in RPMI-1640 medium, supplemented with 10% heat-inactivated FBS (Hyclone, Ogden, UT, USA), 2 mM/ml L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (all from Invitrogen) for 3, 6, and 20 h at 37°C, 5% CO2, in the presence or absence of Lu. longipalpis SGS (0.5 pair/well) or etoposide (20 M). Cells were then cytocentrifuged and stained with Diff-Quick, and pyknotic nuclei were analyzed by light microscopy. In vitro neutrophil infection Peritoneal neutrophils were infected in vitro with L. chagasi promastigotes stationary-phase at a ratio of 1:2 (neutrophil:parasites) in the presence or absence of SGS (0.5 pair/well) in RPMI-1640-supplemented medium. In some experiments, neutrophil infection was performed in the presence of etoposide (20 M). For inhibitory assays, neutrophils were pretreated for 30 min with z-VAD-FMK (100 M) to block caspase activation or preincubated for 1 h with NS-398 (1 M), a COX-2 inhibitor. DMSO (vehicle) 0.4% was used as control. After 20 h, infected neutrophils were centri- www.jleukbio.org 131 Prates et al. Sandfly saliva drives neutrophil apoptosis fuged, supernatants containing noninternalized promastigotes were collected, and medium was replaced by 250 l Schneider medium, supplemented with 20% inactive FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. Infected neutrophils were cultured at 25°C for an additional 3 days. Intracellular load of L. chagasi was estimated by production of proliferating extracellular motile promastigotes in Schneider medium [21]. Quantification of ROS production Intracellular ROS detection in neutrophils cultured at 5 ⫻ 105 cells/well was performed using H2DCFDA fluorescent probe following analyses by FACS, according to the manufacturer’s instructions. For investigation of ROS production, the purified neutrophil population was analyzed by forward- and side-scatter parameters following application of the H2DCFDAFITC probe. Measurement of PGE2 production Supernatants from neutrophil cultures were collected 20 h after incubation with L. chagasi or L. chagasi plus SGS and cleared by centrifugation. PGE2 was measured by the EIA kit from Cayman Chemical. All measurements were performed according to the manufacturer’s instructions. MCP-1/CCL2 measurement Supernatants from neutrophil cultures were collected 20 h after incubation with RPMI medium, SGS, L. chagasi, or L. chagasi plus SGS and cleared by centrifugation. MCP-1 (CCL2) chemokine was measured using the CBA mouse inflammation kit (BD Biosciences), according to the manufacturer’s instructions. Chemotaxis assays Neutrophils were pretreated or not with bindarit propanoic acid (Angelini Farmaceutici; 100 M) for 30 min before incubation with medium, SGS, L. chagasi, or L. chagasi plus SGS, and supernatants were harvested. The culture supernatants were added to the bottom wells of a 96-well chemotaxis microplate ChemoTx system (Neuro Probe, Gaithersburg, MD, USA). Macrophages were obtained 4 days after i.p. injection of 1 ml 3% thioglycolate solution on C57BL/6 mice and ressuspended in RPMI-1640 medium before being added to the top wells (105 cells/well) and incubated for 1.5 h at 37°C under 5% CO2. Following incubation, cells that migrated to the bottom wells were counted on a hemocytometer. Macrophage migration toward RPMI-1640 medium alone (radom chemotaxis) was used as a negative control and toward LPS as a positive control. The chemotaxis indexes were calculated as the ratio of the number of migrated cells toward supernatants taken from L. chagasi-infected or not infected neutrophils cultured in the presence or absence of SGS to the number of cells that migrated to RPMI-1640 medium alone. Statistical analysis The in vitro systems were performed using at least five mice/group. Each experiment was repeated at least three times. Data are reported as mean and se of representative experiments and were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Data distribution from different groups was compared using the Kruskal-Wallis test with Dunn’s multiple comparisons, and comparisons between two groups were explored using the Mann-Whitney test. Differences were considered statistically significant when P ⱕ 0.05. RESULTS Lu. longipalpis SGS induces neutrophil apoptosis Different doses of Lu. longipalpis SGS (0.5–2.0 pairs/well) were capable of inducing apoptosis of neutrophils from C57BL/6 www.jleukbio.org mice (Fig. 1A and C). Such effect was significantly higher than that observed in untreated controls (Fig. 1A and B). The occurrence of apoptosis was similar between the conditions containing diverse doses of SGS (Fig. 1A). We then decided to keep the lowest dose of SGS with biological effect in our model (0.5 pair of salivary gland/well) for further experiments. Neutrophils exhibited markers of apoptosis up to 20 h upon incubation with SGS, such as PS exposure (Fig. 1D) and the pyknotic nuclei (Fig. 1E). At 3 h after stimulus with SGS, indicators had levels similar to those observed in unstimulated cells. Etoposide was used as a positive control to induce neutrophil apoptosis, and its effect was evident at 3 h by annexin-V detection (Fig. 1D) and 20 h by pyknotic nuclei analyses (Fig. 1E). These results confirm the proapoptotic effect of Lu. longipalpis SGS upon murine neutrophils. Our further interest was to explore whether Lu. longipalpis SGS displays a proapoptotic effect on human neutrophils. To address this question, neutrophils obtained from healthy donors were incubated in the presence or absence of SGS or etoposide (Fig. 1F). Strikingly, 3 h after incubation, SGS induced human neutrophil apoptosis (Fig. 1F). At further times (6 and 20 h), this proapoptotic effect was no longer evident by comparison with negative control. Neutrophil apoptosis induced by SGS is caspase-dependent and mediated by FasL To evaluate the mechanisms triggered by Lu. longipalpis saliva to induce neutrophil apoptosis, we incubated C57BL/6 murine neutrophils with z-VAD, a pan-caspase inhibitor, for 30 min before addition of Lu. longipalpis SGS (Fig. 2A). Treatment of neutrophils with z-VAD prevented apoptosis induced by SGS, in contrast to treatment with the vehicle (DMSO) alone (Fig. 2A). Caspase activation can be induced by FasL, a molecule whose expression relates to susceptibility in Leishmania infection [22]. We then assessed FasL expression in neutrophils exposed to Lu. longipalpis SGS, which induced increased expression of FasL in neutrophils concerning intensity/cell (Fig. 2B) and also the percentage of neutrophils expressing FasL (Fig. 2C). Moreover, blockade of FasL prevented neutrophil apoptosis induced by Lu. longiplapis SGS (Fig. 2D). These results indicate that Lu. longipalpis SGS induces neutrophil apoptosis by a mechanism that involves activation of caspases and expression of FasL. Lu. longipalpis SGS proteins induce neutrophil apoptosis To depict initially the composition of the Lu. longipalpis salivary components responsible for the proapoptosis effect on neutrophils, we preincubated SGS with proteinase K before in vitro neutrophil stimulation. We observed a reduction of proapoptotic activity of SGS by incubation with proteinase K (Fig. 3A). This result suggests that apoptosis of neutrophils induced by Lu. longipalpis SGS is mediated by one or more proteic components. Furthermore, as many evidences point out the immunogenicity of sandfly salivary proteins [13, 23, 24], we hypothesized that the Volume 90, September 2011 Journal of Leukocyte Biology 577 132 Figure 1. Effect of Lu. longipalpis SGS on neutrophil apoptosis. (A–E) Neutrophils from C57BL/6 mice were kept unstimulated (–) or stimulated with SGS or etoposide (Etop) 20 M (positive control). (A) Neutrophil apoptosis induced by SGS in different doses was assessed by counting cells with pyknotic nuclei 20 h after stimulation. (B and C) Representative image of inflammatory neutrophils, unstimulated (B) or stimulated with Lu. longipalpis SGS (0.5 pair/well; original magnification, ⫻1000; C). Arrows point to neutrophil pyknotic nuclei. (D and E) Kinetic of neutrophil apoptosis in response to Lu. longipalpis SGS. Three hours and 20 h after stimulation, apoptosis was assessed by flow cytometry after annexin-V staining (D) and by counting cells with pyknotic nuclei (E) on Diff-Quick-stained cytospin preparations. (F) Human neutrophil apoptosis induced by SGS (0.5 pair/well). Data shown are from a single experiment that is representative of three independent experiments. *P ⱕ 0.05; **P ⱕ 0.01, compared with the unstimulated cells. proteic component of the Lu. longipalpis saliva could be targets for the host’s antibodies. To test this possibility, we preincubated the SGS with polled sera from hamsters pre-exposed to Lu. longipalpis bites. Strikingly, preincubation of SGS with specific antiserum completely abrogated induction of neutrophil apoptosis after 20 h in culture (Fig. 3B), reinforcing that components present in Lu. longipalpis saliva with proapoptotic activity are proteins and can be neutralized by antibodies. Effect of Lu. longipalpis SGS in apoptosis and parasite burden of infected neutrophils After determining the proapoptotic effect of Lu. longipalpis SGS, we evaluated whether L. chagasi, the parasite transmitted by this sandfly, can modify this effect in vitro. Analysis of PS exposure on inflammatory neutrophils demonstrated that L. chagasi was also able to induce neutrophil apoptosis (Fig. 4A). Moreover, this effect was exacerbated when neutrophils were coincubated with parasite and saliva (L. chagasi vs. L. chagasi plus SGS: 29.19% vs. 46.39%; Fig. 4A). Neutrophils can act as important host cells for Leishmania [10, 25, 26]. As sandfly saliva exacerbates Leishmania infection [27], we investigated the infection of inflammatory neutrophils with L. 578 Journal of Leukocyte Biology Volume 90, September 2011 chagasi in the presence of Lu. longipalpis SGS in vitro. Saliva increased the viability of L. chagasi inside neutrophils (Fig. 4B). Infection in the presence of etoposide did not enhance parasite burden in neutrophils compared with the control cultures infected with L. chagasi alone (Fig. 4B). Apoptotic neutrophils displayed a high number of parasites (Fig. 4C). To investigate whether neutrophil apoptosis induced by Lu. longipalpis saliva affects this increase of parasite burden in vitro, we pretreated the cultures with z-VAD (Fig. 4D), which abolished the increase in L. chagasi replication induced by SGS (Fig. 4D). COX activation is associated with an increase of Leishmania infection [28]. Herein, we evaluated the role of COX-2, an inflammatory form of COX, in the increase of parasite burden triggered by SGS. NS-398, a COX-2 inhibitor, led to an inhibition of viable parasite number (Fig. 4D) when added to the neutrophil culture before infection. Moreover, PGE2, a product of COX-2, favors intracellular pathogen growth, a phenomenon that could be reverted by treatment with COX-2 inhibitors [29, 30]. Indeed, our experiments show that SGS increased production of PGE2 by Leishmania-infected neutrophils (Fig. 4E). As ROS production is a primarily important microbicidal mechanism from neutrophils, we evaluated the effect of SGS on www.jleukbio.org Prates et al. Sandfly saliva drives neutrophil apoptosis phage recruitment in vitro. We found that supernatants obtained from neutrophil cultures in the presence of L. chagasi could attract macrophages (Fig. 5A) and that Lu. longipalpis saliva induced a synergistic effect (Fig. 5A). Analyses of the MCP-1 (CCL2) revealed that neutrophils incubated with L. chagasi plus SGS produced significantly higher amounts of this chemokine (Fig. 5B). To investigate whether the macrophage recruitment was a result of production of CCL2/MCP-1 induced by L. chagasi plus SGS, we previously treated the neutrophils with bindarit, an inhibitor of CCL2/MCP-1 synthesis, before incubation with SGS, L. chagasi, or both. Treatment with bindarit resulted in total reduction of macrophage chemotaxis (Fig. 5B).Taken together, these results indicate that SGS synergizes with L. chagasi to enhance neutrophil apoptosis, CCL2/ MCP-1 production, and macrophage recruitment. DISCUSSION Figure 2. FasL expression and inhibition of neutrophil apoptosis by zVAD and anti-FasL. (A) Neutrophils from C57BL/6 mice were pretreated with the pan-caspase inhibitor z-VAD (100 M) or with vehicle (DMSO) before incubation with SGS. Twenty hours after incubation, apoptosis was assessed by annexin-V staining. (B and C) FasL expression induced by SGS on neutrophils was analyzed by flow cytometry 20 h after incubation. Results are expressed as the mean fluorescence intensity (MFI) (B) and percentage of FasL-expressing neutrophils on the Gr-1 population (C). (D) Mouse neutrophils were pretreated with neutralizing antibody specific for FasL (␣-FasL; 10 g/ml) or with IgGk1 (10 g/ml). Apoptosis was assessed by annexin-V staining after 20 h. Data shown are from a single experiment representative of three independent experiments. –, Unstimulated (Unst) cells. *P ⱕ 0.05; **P ⱕ 0.01. ROS production by these cells (Fig. 4E). Addition of SGS on the neutrophil cultures induced a partial reduction on ROS production 1 h after infection with L. chagasi (Fig. 4E). In summary, these results suggest that neutrophil apoptosis induced by Lu. longipalpis SGS favors L. chagasi infection by COX-2 activation and PGE2 production, while reducing ROS generation. CCL2/MCP-1 released by L. chagasi-infected neutrophils induces macrophage recruitment We next examined whether supernatans from neutrophils incubated with L. chagasi and SGS are able to induce macro- www.jleukbio.org The present study provides the first evidence that salivary components from a Leishmania vector play a relevant and direct role on neutrophils, which in turn, influence the L. chagasi parasite burden. We found that Lu. longipalpis salivary components induced neutrophil FasL-mediated and caspase-dependent apoptosis, and this event was associated with Leishmania survival inside these cells. Neutrophils are now generally considered an initial target of Leishmania parasites [10, 31]. Significant numbers of neutrophils are present at the parasite inoculation site, as well as in lesions and draining LNs in Leishmania experimentally infected mice [11, 32–35]. Moreover, Lu. longipalpis SGS induces accumulation of neutrophils on an air-pouch model [20]. These experimental data are reinforced by the the fact that massive dermal neutrophilic infiltrates are noted in Lu. longipalpis [13] and Phlebotomus duboscqi bite sites [10], suggesting that accumulation of this cell type may be orchestrated, at least in part, by sandfly saliva constituents. Besides neutrophil recruitment, there are no previous reports about the Figure 3. Inhibition of neutrophil apoptosis after Lu. longipalpis SGS treatment with proteinase K and ␣-saliva serum. Annexin-V staining from C57BL/6 mice neutrophils incubated for 20 h with SGS pretreated with proteinase K (PK; A) or with SGS preincubated for 1 h with anti-Lu. longipalpis saliva serum (B). Data shown are from a single experiment representative of three independent experiments. *P ⱕ 0.05. Volume 90, September 2011 Journal of Leukocyte Biology 579 133 Figure 4. Effect of Lu. longipalpis SGS on neutrophil apoptosis and infection. (A) Inflammatory neutrophils from C57BL/6 mice were kept unstimulated (–) or stimulated with SGS (0.5 pair/ well), L. chagasi (L.c.; 2:1) or SGS ⫹ L. chagasi. After 20 h, apoptosis was assessed by annexin-V staining. (B) In vitro neutrophil infection in the presence of SGS or etoposide (20 M), followed by cultivation at 26°C and viable promastigote counts after 1, 2, and 3 days. (C) Representative image of L. chagasi-infected apoptotic neutrophils stimulated with Lu. longipalpis SGS (0.5 pair/well; original magnification, ⫻1000). Arrows point to infected apoptotic neutrophils. (D) Prior treatment of neutrophils with z-VAD (100 M) and NS-398 (1 M), followed by infection in the presence or absence of SGS. Viable promastigote counts were performed after 3 days. (E) PGE2 levels of supernatants from neutrophils incubated for 20 h with L. chagasi and/or SGS (left side). ROS production by neutrophils cultured with L. chagasi for 1 h in the presence or absence of SGS (right side). Neutrophils were incubated with H2DCFDA, and ROS production was evaluated by flow cytometry. Data shown are from a single experiment representative of three independent experiments. *P ⱕ 0.05; **P ⱕ 0.01. further effects of sandfly saliva on neutrophils. Interestingly, studies performed with tick saliva reveal that the inhibition of critical functions of neutrophils favors the initial survival of spirochetes [36 –38]. Our findings on human neutrophils confirm apoptosis induction by SGS and interestingly, indicate that mice and human neutrophils have a different kinetic of spontaneous and saliva-induced apoptosis. Notably, the apoptosis of human neutrophils induced by Lu. longipalpis SGS also indicates that this mechanism may be important for the pathogenesis of human disease. Indeed, phagocytosis of apoptotic human neutrophils increases parasite burden in macrophages infected with Leishmania amazonensis [28]. It is likely that proteins from SGS trigger neutrophil apoptosis, as reincubation of Lu. longipalpis SGS with proteinase K abrogated its proapoptosis effect. Additionally, antisaliva serum was able of block neutrophil apoptosis. This is particularly interesting, as it reinforces the idea of a host protection mediated by the immune response against sandfly saliva, allowing for the development of an immune response against Leishmania. Interestingly, SGS-induced neutrophil apoptosis was associated with caspases and FasL expression. Previous studies have implicated FasL in neutrophil apoptosis [39]. Likewise, turnover of neutrophils mediated by FasL drives Leishmania major infection [22]. Further studies are necessary to deeply address this observation. Our results demonstrate that SGS increases the neutrophil leishmanial burden by inducing neutrophil apoptosis, as inhibition of apoptosis by z-VAD reduced the viable parasite numbers in vitro. Indeed, treatment with z-VAD blocks lymphocyte 580 Journal of Leukocyte Biology Volume 90, September 2011 apoptosis and increases in vitro and in vivo resistance to Trypanosoma cruzi infection [30, 40]. van Zandbergen and colleagues [12] have proposed that infected apoptotic neutrophils can serve as “Trojan horses” for Leishmania. Alternatively, uptake of parasites egressing from dying neutrophils in an anti-inflammatory environment created by the phagocytosis of these cells, per se, could favor the infection (“Trojan rabbit” strategy) [41]. Our findings that Lu. longipalpis SGS could favor neutrophil apoptosis and infection by L. chagasi seem to give support to either of these two proposed hypotheses. We found that neutrophil infection in the presence of SGS induced PGE2 release, but was decreased in the presence of COX-2 inhibitor NS-398, indicating the participation of COX-2 products in parasite survival. Indeed, PGE2, a major product from COX-2, facilitates Leishmania infection by deactivating macrophage microbicidal functions [19, 28 –30]. Moreover, addition of exogenous PGE2 to macrophage cultures induces a marked enhancement of Leishmania infection [19, 42]. Exposure of neutrophils to SGS caused a marked reduction of ROS production, which is a primarily important microbicidal mechanism of neutrophils. In this regard, Lu. longipalpis salivary proteins could be contributing to deactivation of the neutrophil inflammatory response, favoring the early steps of Leishmania infection. Taken together, our data suggest that the presence of sandfly SGS drives an anti-inflammatory response in L. chagasi-infected neutrophils by initially reducing ROS production, favoring the parasite survival. Furthermore, SGS could be triggering neutrophil deactivation through induction of apoptosis, activation of COX-2, and PGE2 production by these cells. L. major promastigotes drive a selective fusion of azuro- www.jleukbio.org 134 Prates et al. Sandfly saliva drives neutrophil apoptosis tween vector saliva and neutrophils in innate immunity to Leishmania infection. AUTHORSHIP D.B.P., T.A-S., B.B.A., M.B-N., V.M.B., and A.B. conceived of and designed the experiments. D.B.P., T.A-S., N.F.L., J.C., B.B.A., L.A., and J.F-C. performed the experiments. D.B.P., T.A-S., J.C., L.A., M.B-N., V.M.B., and A.B. analyzed the data. J.C.M., M.B-N., V.M.B., and A.B. contributed reagents/materials/analysis tools. D.B.P., T.A-S., B.B.A., M.B-N., and V.M.B. wrote the paper. D.B.P., T.A-S., B.B.A., P.T.B., G.A.D., C.B., M.B-N., V.M.B., and A.B. participated in critical discussion of the manuscript. Figure 5. Macrophage recruitment and CCL2/MCP-1 release by L. chagasi-infected neutrophils in the presence of Lu. longipalpis SGS. (A) Macrophages were allowed to migrate toward supernatants from neutrophils infected or not with L. chagasi in the presence or absence of SGS (white bars), as described in Materials and Methods. Migration toward supernatants from bindarit-pretreated neutrophils (black bars). Following incubation, the migrated macrophages were counted, and the chemotatic index was calculated. Crtl, Negative control of radom chemotaxis. (B) CCL2/MCP-1 production (white bars) in the supernatants of neutrophil cultures after 20 h and its inhibition by bindarit pretreatment (black bars). Data are representative of two independent experiments performed in triplicate for each sample. *P ⱕ 0.05; #P ⱕ 0.05, compared with no bindarit-treated neutrophils. ACKNOWLEDGMENTS This work was supported by CNPq, Instituto Nacional de Ciência e Tecnologia de Investigação em Imunologia (iii-INCT), and Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB). D.B.P., T.A-S., N.F.L., and L.A. are recipients of a CNPq fellowship. J.F-C. is the recipient of a CAPES fellowship. P.T.B., G.A.D., C.B., M.B-N., V.M.B., and A.B. are senior investigators from CNPq. We thank Edvaldo Passos for technical assistance with the insect colony. REFERENCES philic granules into parasite-containing phagosomes in human neutrophils [43]. It remains to be elucidated whether, in the present system, SGS modulates neutrophil granule mobilization and contributes to early L. chagasi survival. Macrophages are the preferential host cells for Leishmania, and the recruitment of these cells could provide safe havens for the parasite [31]. Neutrophils infected by L. major produce chemokines such as MIP-1 [12, 44], and sandfly SGS leads to increased expression of the macrophage chemokine MCP-1 at the site of injection [20], leading to macrophage recruitment. We have shown here that neutrophils infected with L. chagasi in the presence of SGS displayed higher MCP-1 production, corroborating with macrophage recruitment. This result was reinforced with the use of bindarit, an original indazolic derivative that has been shown the ability to inhibit CCL2/MCP-1 synthesis [45]. As a matter of fact, L. chagasi-infected neutrophil supernatants are able to recruit mouse macrophages, even though they did not induce significant MCP-1 production, which suggests that other chemotatic factors could be implicated in this event. A direct chemotatic activity of sandfly saliva has been described with several experimental models [13, 20, 46]. Herein, we also report an indirect chemotactic effect of SGS by inducing chemokine production by neutrophils. In summary, our data demonstrate that Lu. longipalpis saliva orchestrates FasL- and caspase-dependent apoptosis of neutrophils. 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O., Oliveira, F., Clarencio, J., Noronha, A., Barral, A., Brodskyn, C., de Oliveira, C. I. (2005) Toward a novel experimental model of infection to study American cutaneous leishmaniasis caused by Leishmania braziliensis. Infect. Immun. 73, 5827–5834. Lima, G. M., Vallochi, A. L., Silva, U. R., Bevilacqua, E. M., Kiffer, M. M., Abrahamsohn, I. A. (1998) The role of polymorphonuclear leukocytes in the resistance to cutaneous Leishmaniasis. Immunol. Lett. 64, 145–151. Pompeu, M. L., Freitas, L. A., Santos, M. L., Khouri, M., Barral-Netto, M. (1991) Granulocytes in the inflammatory process of BALB/c mice infected by Leishmania amazonensis. A quantitative approach. Acta Trop. 48, 185–193. Guo, X., Booth, C. J., Paley, M. A., Wang, X., DePonte, K., Fikrig, E., Narasimhan, S., Montgomery, R. R. (2009) Inhibition of neutrophil function by two tick salivary proteins. Infect. Immun. 77, 2320 –2329. Montgomery, R. R., Lusitani, D., De Boisfleury Chevance, A., Malawista, S. E. (2004) Tick saliva reduces adherence and area of human neutrophils. Infect. Immun. 72, 2989 –2994. Ribeiro, J. M., Weis, J. J., Telford III, S. R. (1990) Saliva of the tick Ixodes dammini inhibits neutrophil function. Exp. Parasitol. 70, 382–388. Borges, V. M., Falcao, H., Leite-Junior, J. H., Alvim, L., Teixeira, G. P., Russo, M., Nobrega, A. F., Lopes, M. F., Rocco, P. M., Davidson, W. F., Linden, R., Yagita, H., Zin, W. A., DosReis, G. A. (2001) Fas ligand triggers pulmonary silicosis. J. Exp. Med. 194, 155–164. Silva, E. M., Guillermo, L. V., Ribeiro-Gomes, F. L., De Meis, J., Nunes, M. P., Senra, J. F., Soares, M. B., DosReis, G. A., Lopes, M. F. (2007) Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection. Eur. J. Immunol. 37, 738 – 746. Ritter, U., Frischknecht, F., van Zandbergen, G. (2009) Are neutrophils important host cells for Leishmania parasites? Trends Parasitol. 25, 505– 510. Lonardoni, M. V., Barbieri, C. L., Russo, M., Jancar, S. (1994) Modulation of Leishmania (L.) amazonensis growth in cultured mouse macrophages by prostaglandins and platelet activating factor. Mediators Inflamm. 3, 137–141. Mollinedo, F., Janssen, H., de la Iglesia-Vicente, J., Villa-Pulgarin, J. A., Calafat, J. (2010) Selective fusion of azurophilic granules with Leishmaniacontaining phagosomes in human neutrophils. J. Biol. Chem. 285, 34528 – 34536. Rupp, J., Pfleiderer, L., Jugert, C., Moeller, S., Klinger, M., Dalhoff, K., Solbach, W., Stenger, S., Laskay, T., van Zandbergen, G. (2009) Chlamydia pneumoniae hides inside apoptotic neutrophils to silently infect and propagate in macrophages. PLoS ONE 4, e6020. Mirolo, M., Fabbri, M., Sironi, M., Vecchi, A., Guglielmotti, A., Mangano, G., Biondi, G., Locati, M., Mantovani, A. (2008) Impact of the anti-inflammatory agent bindarit on the chemokinome: selective inhibition of the monocyte chemotactic proteins. Eur. Cytokine Netw. 19, 119 –122. Monteiro, M. C., Lima, H. C., Souza, A. A., Titus, R. G., Romao, P. R., Cunha, F. Q. (2007) Effect of Lutzomyia longipalpis salivary gland extracts on leukocyte migration induced by Leishmania major. Am. J. Trop. Med. Hyg. 76, 88 –94. KEY WORDS: Leishmania chagasi 䡠 sand fly 䡠 cell death 䡠 FasL 䡠 chemotaxis www.jleukbio.org 136 The Journal of Immunology Heme Impairs Prostaglandin E2 and TGF-b Production by Human Mononuclear Cells via Cu/Zn Superoxide Dismutase: Insight into the Pathogenesis of Severe Malaria Bruno B. Andrade,*,†,1 Théo Araújo-Santos,*,† Nı́vea F. Luz,*,† Ricardo Khouri,‡ Marcelo T. Bozza,x Luı́s M. A. Camargo,{,‖ Aldina Barral,*,†,# Valéria M. Borges,*,# and Manoel Barral-Netto*,†,# S evere malaria is a highly lethal condition and a major health threat in many tropical countries. Multiple factors have been implicated in the pathogenesis of the severe complications of this condition, such as uncontrolled cytokine production (1, 2), hemolysis (3), and erythropoiesis suppression (4). Severe malaria was firstly described as originating from Plasmodium falciparum infection (5), but severe cases, including those with lethal outcomes, have also been observed from Plasmodium vivax infections (6–8). One of the major factors thought to be involved in sustaining systemic inflammation is the release of free heme, as a consequence of *Centro de Pesquisas Gonçalo Moniz (Fundação Oswaldo Cruz); †Faculdade de Medicina da Bahia, Universidade Federal da Bahia, Salvador; {Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade de São Paulo; #Instituto de Investigação em Imunologia (iii), Instituto Nacional de Ciência e Tecnologia, São Paulo; ‖Faculdade de Medicina, Faculdade São Lucas, Porto Velho; xDepartamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and ‡Rega Institute, Katholieke Universiteit, Leuven, Belgium 1 Current address: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. Received for publication December 29, 2009. Accepted for publication May 13, 2010. This work was supported by Financiadora de Estudos e Projetos (Grant 010409605)/ Fundo Nacional de Desenvolvimento Cientifico e Tecnológico Amazônia. B.B.A., T.A.S., and N.F.L. received fellowships from the Brazilian National Research Council (Conselho Nacional de Pesquisa e Tecnologia). M.T.B., V.M.B., A.B., and M.B.-N. are senior investigators from the Conselho Nacional de Pesquisa e Tecnologia. Address correspondence and reprint requests to Dr. Manoel Barral-Netto, Centro de Pesquisas Gonçalo Moniz (Fundação Oswaldo Cruz), Rua Waldemar Falcão, 121, Salvador, Bahia, Brazil, CEP 40295-001. E-mail address: [email protected] Abbreviations used in this paper: 7-AAD, 7-aminoactinomycin D; A, asymptomatic; ALT, alanine aminotransferase; CoPPIX, cobalt protoporphyrin IX; CRP, C-reactive protein; DETC, diethyldithiocarbamate; Hb, hemoglobin; HO-1, heme oxygenase-1; M, mild; NAC, N-acetyl-L-cysteine; NI, noninfected individual; PPIX, protoporphyrin IX; ROS, reactive oxygen species; S, severe; siRNA, small interfering RNA; SnPPIX, Tin protoporphyrin IX; SOD-1, Cu/Zn superoxide dismutase. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0904179 hemolysis inherent to the life cycle of Plasmodium within RBCs (9). Recently, heme has been implicated in the pathogenesis of severe forms of malaria in mice (10, 11). Under homeostasis, the heme released from hemoproteins such as cell-free hemoglobin (Hb) is scavenged by plasma proteins such as hemopexin or albumin as well as by lipoproteins (12). However, these proteins can be depleted during severe hemolytic conditions, such as associated with Plasmodium infection (13). This leads to the accumulation of free Hb tetramers in the plasma (14), which dissociate spontaneously into dimers. In the presence of reactive oxygen species (ROS) or other free radicals, cell-free Hb dimers are readily oxidized into methemoglobin, releasing their heme prosthetic groups (12). As a consequence, in malaria and other hemolytic disorders, the concentrations of heme can reach levels of up to 50 mM in the bloodstream (15), which can trigger an intense oxidative burst and unspecific tissue damage (11). Moreover, a crystal form of heme molecules produced by Plasmodium sp., and referred to as hemozoin, also acts as a proinflammatory agonist and thus could be associated with the development of severe forms of malaria (16–18). Hemozoin inhibits PGE2 production in both mice (19) and humans (20, 21), and there is an inverse relationship between PGE2 and blood mononuclear cell cyclooxygenase-2 with disease severity in children with P. falciparum malaria (22). Until now there is no clear description of the effect of free heme on the PGE2 production. During malaria infection, superoxide anions are thought to be the main form of ROS produced (23). In this context, the antioxidant enzyme Cu/Zn superoxide dismutase (SOD-1) is activated and may display an important role in the pathological oxidative injury. Notwithstanding, SOD-1 has been linked to an increased inflammatory activity by amplifying TNF-a production on macrophages (24). In addition, overexpression of SOD-1 increases NF-kB–related rapid responses, such as immune response and antiapoptosis factors (25). Therefore, studies have correlated SOD-1 activity with Downloaded from http://jimmunol.org/ by guest on October 26, 2012 In many hemolytic disorders, such as malaria, the release of free heme has been involved in the triggering of oxidative stress and tissue damage. Patients presenting with severe forms of malaria commonly have impaired regulatory responses. Although intriguing, there is scarce data about the involvement of heme on the regulation of immune responses. In this study, we investigated the relation of free heme and the suppression of anti-inflammatory mediators such as PGE2 and TGF-b in human vivax malaria. Patients with severe disease presented higher hemolysis and higher plasma concentrations of Cu/Zn superoxide dismutase (SOD-1) and lower concentrations of PGE2 and TGF-b than those with mild disease. In addition, there was a positive correlation between SOD-1 concentrations and plasma levels of TNF-a. During antimalaria treatment, the concentrations of plasma SOD-1 reduced whereas PGE2 and TGF-b increased in the individuals severely ill. Using an in vitro model with human mononuclear cells, we demonstrated that the heme effect on the impairment of the production of PGE2 and TGF-b partially involves heme binding to CD14 and depends on the production of SOD-1. Aside from furthering the current knowledge about the pathogenesis of vivax malaria, the present results may represent a general mechanism for hemolytic diseases and could be useful for future studies of therapeutic approaches. The Journal of Immunology, 2010, 185: 1196–1204. 137 Published March 28, 2012, doi:10.4049/jimmunol.1103072 The Journal of Immunology Heme Oxygenase-1 Promotes the Persistence of Leishmania chagasi Infection Nı́vea F. Luz,*,† Bruno B. Andrade,‡ Daniel F. Feijó,x Théo Araújo-Santos,*,† Graziele Q. Carvalho,*,† Daniela Andrade,*,† Daniel R. Abánades,*,† Enaldo V. Melo,{ Angela M. Silva,{ Cláudia I. Brodskyn,*,†,‖ Manoel Barral-Netto,*,†,‖ Aldina Barral,*,†,‖ Rodrigo P. Soares,# Roque P. Almeida,{,‖ Marcelo T. Bozza,x and Valéria M. Borges*,†,‖ V isceral leishmaniasis (VL) continues to be a major health threat worldwide and is classified as one of the most neglected diseases by the World Health Organization. VL is a chronic infection clinically characterized by progressive fever, weight loss, splenomegaly, hepatomegaly, anemia, and spon- *Centro de Pesquisas Gonçalo Moniz/Fundação Oswaldo Cruz, Salvador 40295-001, Brazil; †Universidade Federal da Bahia, Salvador 40110-060, Brazil; ‡Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; xDepartamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; {Department of Medicine, University Hospital, Universidade Federal de Sergipe, Aracaju 49010-390, Brazil; ‖Instituto Nacional de Ciência e Tecnologia de Investigação em Imunologia, Salvador, Bahia 40110-100, Brazil; and #Centro de Pesquisas René Rachou/Fundação Oswaldo Cruz, Belo Horizonte 30190-002, Brazil Received for publication October 27, 2011. Accepted for publication March 1, 2012. This work was supported by Fundação de Amparo a Pesquisa do Estado da Bahia, Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq), and Instituto Nacional de Ciência e Tecnologia de Investigação em Imunologia. N.F.L., D.F.F, T.A.-S., and G.Q.C. are recipients of CNPq fellowships. D.A. receives a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior. C.I.B., R.P.S., M.B.-N., A.B., R.P.A., M.T.B., and V.M.B. are senior investigators from CNPq. The work of B.B.A. is supported by the intramural research program of the National Institute for Allergy and Infectious Diseases, National Institutes of Health. Address correspondence and reprint requests to Dr. Valéria M. Borges, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão, 121, Candeal, Salvador, Bahia 40295-001, Brazil. E-mail address: vborges@bahia. fiocruz.br The online version of this article contains supplemental material. Abbreviations used in this article: BMM, bone marrow-derived macrophage; CoPP, cobalt protoporphyrin IX; DHE, dihydroethidium; HC, healthy control; HO-1, heme oxygenase-1; LPG, lipophosphoglycan; PPARg, peroxisome proliferator-activated receptor g; PTX, pentoxifylline; ROC, receiver-operator characteristic; ROS, reactive oxygen species; SOD-1, Cu/Zn superoxide dismutase; VL, visceral leishmaniasis; WT, wild-type. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103072 taneous bleeding associated with marked inflammatory imbalance (1). The hallmark of this disease is thought to be a lack of cellular immune responses against the parasite and high systemic levels of IFN-g and IL-10 (2). The New World Leishmania infantum chagasi is the major species implicated in the VL in Brazil. Leishmania parasites are obligate intracellular protozoa that replicate preferentially inside macrophages (3). It is well known that L. chagasi is able to evade pro-oxidative responses and other macrophage effectors mechanisms (4), possibly hampering the activation of adaptive immune responses against infection (5). During parasite–host interactions, complex signaling pathways are triggered by the recognition of key molecules from parasite (4). In this context, lipophosphoglycan (LPG), a glycoconjugate expressed on the surface of Leishmania parasites and TLR2 agonist (6, 7), has been implicated in the modulation of a wide range of innate immune functions. Those may include resistance to complement, attachment and entry into macrophages, protection against proteolytic damage within acidic vacuoles (8), inhibition of phagosomal maturation (9), modulation of NO and IL-12 production (10–13), inhibition of protein kinase C (14), induction of neutrophil extracellular traps (15), and induction of protein kinase R (16). However, specific aspects of how the parasites regulate some protective responses are still unknown. Moreover, it is not fully understood whether LPG from Leishmania is the major regulator of the effectors pathways associated with the protective responses against this protozoan. Excess of heme is very hazardous for the cells, and we have previously shown that heme suppresses some anti-inflammatory mediators in human malaria caused by Plasmodium vivax (17). Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that Downloaded from www.jimmunol.org on March 28, 2012 Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-a and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach. The Journal of Immunology, 2012, 188: 000–000. 138 642 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 105(5): 642-648, August 2010 Control of Mycobacterium fortuitum and Mycobacterium intracellulare infections with respect to distinct granuloma formations in livers of BALB/c mice Tânia Regina Marques da Silva, Antonio Luis de Oliveira Almeida Petersen, Theo de Araújo Santos, Taís Fontoura de Almeida, Luiz Antônio Rodrigues de Freitas, Patrícia Sampaio Tavares Veras/+ Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz-Fiocruz, Rua Waldemar Falcão 121, 40296-710 Salvador, BA, Brasil Mycobacterium fortuitum is a rapidly growing nontuberculous Mycobacterium that can cause a range of diseases in humans. Complications from M. fortuitum infection have been associated with numerous surgical procedures. A protective immune response against pathogenic mycobacterial infections is dependent on the granuloma formation. Within the granuloma, the macrophage effector response can inhibit bacterial replication and mediate the intracellular killing of bacteria. The granulomatous responses of BALB/c mice to rapidly and slowly growing mycobacteria were assessed in vivo and the bacterial loads in spleens and livers from M. fortuitum and Mycobacterium intracellulare-infected mice, as well as the number and size of granulomas in liver sections, were quantified. Bacterial loads were found to be approximately two times lower in M. fortuitum-infected mice than in M. intracellulareinfected mice and M. fortuitum-infected mice presented fewer granulomas compared to M. intracellulare-infected mice. These granulomas were characterized by the presence of Mac-1+ and CD4+ cells. Additionally, IFN-γ mRNA expression was higher in the livers of M. fortuitum-infected mice than in those of M. intracellulare-infected mice. These data clearly show that mice are more capable of controlling an infection with M. fortuitum than M. intracellulare. This capacity is likely related to distinct granuloma formations in mice infected with M. fortuitum but not with M. intracellulare. Key words: Mycobacterium fortuitum - Mycobacterium intracellulare - granuloma - liver - control of infection Nontuberculous mycobacteria (NTM) include different species of the genus Mycobacterium that do not belong to the Mycobacterium tuberculosis complex. These include both slowly growing [e.g., Mycobacterium avium-intracellulare (MAI)] and rapidly growing (e.g., Mycobacterium fortuitum and Mycobacterium abscessus) species (Runyon 1959). NTM are human opportunistic pathogens and are predominantly acquired from the environment. A large number of NTM species have been recovered from soil, household dust, water, dairy products, cold-blooded animals, vegetation and human faeces (Ho et al. 2006). These species can also colonize surgical equipment and materials, such as endoscopes and solutions (Brown-Elliott & Wallace 2005). In humans, NTM are organisms that belong to a heterogeneous group in which each species of bacteria should be studied separately (Alvarez-Uria 2010). These pathogens can cause a range of diseases affecting a variety of tissues, including the lungs, lymph nodes, skin and soft and skeletal tissue. These diseases can also affect the genitourinary systems and cause disseminated infections (Ho et al. 2006, Griffith et al. 2007, Jarzembowski Financial support: CNPq (306672/2008-1) + Corresponding author: [email protected] Received 14 January 2010 Accepted 15 June 2010 online | memorias.ioc.fiocruz.br & Young 2008). MAI is primarily a pulmonary pathogen and is the NTM species most commonly associated with human disease (Griffith et al. 2007). Inhalation of this bacterium may cause pulmonary disease, whereas the ingestion of contaminated water may cause a disseminated disease. A cutaneous manifestation can be attributed to direct inoculation, direct contact or disseminated disease (Weitzul et al. 2000). Infections caused by rapidly growing NTM including M. fortuitum can appear after surgical procedures, such as liposuction, silicone injection and breast implantation, or after intravenous catheter insertion, exposure to prosthetic material and pacemaker placement (Sungkanuparph et al. 2003, Palwade et al. 2006, Uslan et al. 2006). There is still no defined optimal treatment for NTM infections because these organisms are resistant to the standard antituberculous agents. In addition, susceptibility to anti-mycobacterial agents varies across different NTM species (ATS 1997). A protective immune response against pathogenic mycobacterial infections depends on the ability of individuals to form organ granulomas. During infection, mycobacteria induce the formation of these organized immune complexes of differentiated macrophages, lymphocytes and other cells, which are critical for the maintenance of the granuloma architecture and for the restriction of the infection. In the centre of the granuloma, macrophages produce a response that can effectively prevent the replication of bacteria and/or mediate the killing of the intracellular pathogen. On the other hand, compromised granuloma formation is accompanied by dissemination. In addition, the course of the infection in individuals that
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